1: Mol Cell Proteomics. 2005 Oct 16; [Epub ahead of print] New data-analysis and mining approaches identify unique proteome and transcriptome markers of susceptibility to autoimmune diabetes. Gerling IC, Singh S, Lenchik NI, Marshall DR, Wu J. Medicine, University of Tennessee Health Science Center, Memphis, TN 38104. Non-obese diabetic (NOD) mice spontaneously develop autoimmunity to the insulin producing beta-cells leading to insulin dependent diabetes. In this study we have developed and used new data analysis and mining approached on combined proteome and trascriptome (molecular phenotype) data, to define pathways affected by abnormalities in peripheral leukocytes of young NOD female mice. Cells were collected before mice show signs of autoimmunity (age 2-4 weeks). We extracted both protein and RNA from NOD and C57BL/6 control mice to conduct both proteome analysis by 2 Dimensional (2D) gel electrophoresis and transcriptome analysis on Affymetrix expression arrays. We developed a new approach to analyze the 2D-gel proteome data, that included 2-way ANOVA, cluster analysis and Principal Component Analysis (PCA). Lists of differentially expressed proteins and transcripts were subjected to pathway analysis using a commercial service. From the list of 24 proteins differentially expressed between strains we identified two highly significant and interconnected networks centered around oncogenes (Myc and Mycn) and apoptosis related genes (Bcl2 and Casp3). The 273 genes with significant strain differences in RNA expression levels created six interconnected networks with a significant overrepresentation of genes related to cancer, cell cycle, and cell death. They contained many of the same genes found in the proteome networks (including Myc and Mycn). The combination of the eight, highly significant, networks created one large network of 272 genes of which 82 had differential expression between strains either at the protein or the RNA level. We conclude that new proteome data analysis strategies and combined information from proteome and transcriptome can enhance the insights gained from either type of data alone. The overall systems biology of prediabetic NOD mice points towards abnormalities in regulation of the opposing processes of cell renewal and cell death, even before there are any clear signatures of immune system activation. PMID: 16227630 [PubMed - as supplied by publisher] --------------------------------------------------------------- 2: Neuropathology. 2005 Sep;25(3):178-87. Tumor cell-associated neuropilin-1 and vascular endothelial growth factor expression as determinants of tumor growth in neuroblastoma. Marcus K, Johnson M, Adam RM, O'Reilly MS, Donovan M, Atala A, Freeman MR, Soker S. Department of Radiation Oncology, Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA. We sought to characterize the expression of vascular endothelial growth factor (VEGF) and its receptors in neuroblastoma (NBL) and to correlate the results with N-myc (MYCN) expression and in vivo growth of these tumors. Two representative human-derived NBL cell lines, SK-N-AS (AS) with low and SK-N-DZ (DZ) with a high MYCN copy number, were used for the study. We examined their proliferation, VEGF and VEGF receptor expression in vitro and xenograft tumor growth in vivo. In parallel, human NBL specimens were analyzed for expression of VEGF and neuropilin-1 (NRP-1). DZ cells exhibited a 4-fold higher proliferation rate than AS. In contrast, VEGF protein expression was significantly higher in AS cells. NRP-1 was the only VEGF receptor produced in AS and DZ cells in vitro and in vivo. Both AS and DZ cells formed tumors in athymic mice but AS tumors grew 3.5 times larger than DZ tumors and had larger diameter tumor vessels. VEGF and NRP-1 expression was also demonstrated in human NBL specimens. Our studies indicate that VEGF and VEGF receptor expression in NBL tumor cells are associated with tumor growth and that angiogenic factors may serve as a biological marker together with already established MYCN amplification. PMID: 16193833 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: Oncogene. 2005 Aug 22; [Epub ahead of print] Combined microarray analysis of small cell lung cancer reveals altered apoptotic balance and distinct expression signatures of MYC family gene amplification. Kim YH, Girard L, Giacomini CP, Wang P, Hernandez-Boussard T, Tibshirani R, Minna JD, Pollack JR. 1Department of Pathology, Stanford University, Stanford, CA, USA. DNA amplifications and deletions frequently contribute to the development and progression of lung cancer. To identify such novel alterations in small cell lung cancer (SCLC), we performed comparative genomic hybridization on a set of 24 SCLC cell lines, using cDNA microarrays representing approximately 22 000 human genes (providing an average mapping resolution of <70 kb). We identified localized DNA amplifications corresponding to oncogenes known to be amplified in SCLC, including MYC (8q24), MYCN (2p24) and MYCL1 (1p34). Additional highly localized DNA amplifications suggested candidate oncogenes not previously identified as amplified in SCLC, including the antiapoptotic genes TNFRSF4 (1p36), DAD1 (14q11), BCL2L1 (20q11) and BCL2L2 (14q11). Likewise, newly discovered PCR-validated homozygous deletions suggested candidate tumor-suppressor genes, including the proapoptotic genes MAPK10 (4q21) and TNFRSF6 (10q23). To characterize the effect of DNA amplification on gene expression patterns, we performed expression profiling using the same microarray platform. Among our findings, we identified sets of genes whose expression correlated with MYC, MYCN or MYCL1 amplification, with surprisingly little overlap among gene sets. While both MYC and MYCN amplification were associated with increased and decreased expression of known MYC upregulated and downregulated targets, respectively, MYCL1 amplification was associated only with the latter. Our findings support a role of altered apoptotic balance in the pathogenesis of SCLC, and suggest that MYC family genes might affect oncogenesis through distinct sets of targets, in particular implicating the importance of transcriptional repression.Oncogene advance online publication, 22 August 2005; doi:10.1038/sj.onc.1208997. PMID: 16116477 [PubMed - as supplied by publisher] --------------------------------------------------------------- 4: J Clin Oncol. 2005 Sep 20;23(27):6474-80. Epub 2005 Aug 22. Comment in: J Clin Oncol. 2005 Sep 20;23(27):6443-4. Favorable prognosis for patients 12 to 18 months of age with stage 4 nonamplified MYCN neuroblastoma: a Children's Cancer Group Study. Schmidt ML, Lal A, Seeger RC, Maris JM, Shimada H, O'Leary M, Gerbing RB, Matthay KK. Department of Pediatrics, University of Illinois at Chicago College of Medicine, Chicago, IL 60612, USA. mls3@uic.edu PURPOSE: The long-term survival of children between age 12 and 24 months with stage 4 neuroblastoma and nonamplified MYCN (MYCN-NA) has not been defined previously. PATIENTS AND METHODS: Survival for stage 4 MYCN-NA neuroblastoma patients enrolled onto Children's Cancer Group (CCG) protocols 321P2 (1986 to 1991) and 3891 (1991 to 1996) was analyzed. Treatment consisted of intensive alkylator-based induction chemotherapy with or without autologous bone marrow transplantation (ABMT) with or without 13 cis-retinoic acid. Survival was analyzed by age strata less than 12, 12 to 18, 18 to 24, and more than 24 months at diagnosis. Patients younger than 12 months were treated on the moderate-intensity CCG protocol 3881. RESULTS: Forty-three patients with stage 4 MYCN-NA disease enrolled onto CCG-321P2 (n = 17) or CCG-3891 (n = 26) were between 12 and 24 months of age at diagnosis. After a median follow-up of 94 months (range, 4 to 140 months), the 6-year event-free survival (EFS) for the 12- to 18-month age group was superior to that of the 18- to 24-month age group (74% +/- 8% v 31% +/- 12%; P = .008). The EFS for children older than 24 months with stage 4 MYCN-NA neuroblastoma was 23% +/- 3%, and for children younger than 12 months was 92% +/- 3%. CONCLUSION: Children diagnosed with stage 4 MYCN-NA neuroblastoma in the second year of life form a transitional group between infants and older children in terms of prognosis. Patients between 12 and 18 months of age have significantly better long-term survival than that of older children treated with intensive chemotherapy with or without ABMT. These patients may not benefit from additional intensification of therapy beyond that provided in earlier clinical trials and may even maintain this high survival rate with less intensive therapy. Publication Types: Clinical Trial Randomized Controlled Trial PMID: 16116154 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: J Clin Oncol. 2005 Sep 20;23(27):6466-73. Epub 2005 Aug 22. Comment in: J Clin Oncol. 2005 Sep 20;23(27):6443-4. Hyperdiploidy plus nonamplified MYCN confers a favorable prognosis in children 12 to 18 months old with disseminated neuroblastoma: a Pediatric Oncology Group study. George RE, London WB, Cohn SL, Maris JM, Kretschmar C, Diller L, Brodeur GM, Castleberry RP, Look AT. Department of Pediatric Hematology and Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA. rani_george@dfci.harvard.edu PURPOSE: To determine predictive strength of tumor cell ploidy and MYCN gene amplification on survival of children older than 12 months with disseminated neuroblastoma (NB). PATIENTS AND METHODS: Of 648 children with stage D NB enrolled onto the Pediatric Oncology Group NB Biology Study 9047 (1990-2000), 560 children were assessable for ploidy and MYCN amplification. Treatment of patients older than 12 months varied; most receiving high-dose chemotherapy with stem-cell rescue. Infants received standard chemotherapy, depending on MYCN status and ploidy. RESULTS: Among stage D MYCN-amplified patients, 4-year event-free survival (EFS) +/- SE had no prognostic significance for tumor cell ploidy for patients either younger than 12 months or > or = 12 months old. However, among stage D nonamplified-MYCN patients, 4-year EFS for those with tumor hyperdiploidy (DNA index [DI] > 1) was clearly superior to those with diploidy (DI < or = 1): younger than 12 months, 83.7% +/- 4.4% (n = 87) versus 46.2% +/- 13.8% (n = 13; P = .0003); and for 12- to 24-month-old children, 72.7% +/- 10.2% (n = 22) versus 26.7% +/- 13.2% (n = 16; P = .0092). Further analysis suggested better prognoses in the 12- to 18-month-old subgroup with hyperdiploid tumors (4-year EFS, 92.9% +/- 7.2%) compared with the 19- to 24-month-old subgroup (4-year EFS, 37.5% +/- 21.0%; P = .0037). In children older than 24 months, outcome was dire (< 20% long-term survival), regardless of ploidy or MYCN status. CONCLUSION: Children 12 to 18 months old with metastatic NB had favorable outcomes with high-dose therapy if their tumors were hyperdiploid and lacked MYCN amplification. This subgroup may respond well to contemporary chemotherapy, and could be spared intensive myeloablative therapy with stem-cell rescue. Publication Types: Clinical Trial Randomized Controlled Trial PMID: 16116152 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: Curr Cancer Drug Targets. 2005 Jun;5(4):273-83. The MYCN oncogene as a specific and selective drug target for peripheral and central nervous system tumors. Pession A, Tonelli R. Department of Pediatrics, University of Bologna, S. Orsola Hospital, Bologna, Italy. pession@med.unibo.it MYCN belongs to the MYC family of proto-oncogenes, which encode for transcription factors of the basic-helix-loop-helix-zipper (bHLHZ) class and is fundamental in the development of the peripheral and central nervous systems (PNS and CNS). While Myc is ubiquitous, MYCN has a very restricted expression pattern: it is mainly expressed during embryonic development, but then becomes downregulated, while in adults it is usually detected in B-cell development. Identification of selective inhibitors of MYCN and its mRNA and protein could be important for the development of more specific, effective and less toxic therapeutic agents for tumors of the PNS and CNS. In children, the most common tumors of the PNS and CNS are neuroblastomas and medulloblastomas, respectively. About 30% of neuroblastoma (NB) tumors present MYCN amplification/over-expression, which is associated with rapid progression and poor prognosis. N-Myc is essential during neurogenesis for the rapid expansion of progenitor cells in the brain. MYCN amplification and over-expression has been reported in medulloblastoma, and especially in the desmoplastic type. Other tumors associated with MYCN overexpression include retinoblastoma, small cell lung carcinoma, glioblastoma and certain embryonal tumors. A cell-based, N-Myc-dependent luciferase reporter gene assay to identify specific N-Myc small-molecule inhibitors has allowed identification of five compounds showing significant activity. Antisense oligodeoxynucleotides have been shown to inhibit N-Myc production and anti-tumoral activity in vitro and in vivo for NB. Peptide nucleic acids (PNA), which belong to the most recent (third) generation of nucleic acid therapeutics, form highly stable duplexes with DNA and RNA, and are resistant to degradation by nucleases and proteases. Encouraging results have been reported utilizing a PNA-based antisense strategy for inhibition of N-Myc expression in neuroblastoma. Publication Types: Review Review, Tutorial PMID: 15975048 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Clin Cancer Res. 2005 Jun 15;11(12):4321-30. Comment in: Clin Cancer Res. 2005 Jun 15;11(12):4278-81. Disruption of cooperation between Ras and MycN in human neuroblastoma cells promotes growth arrest. Yaari S, Jacob-Hirsch J, Amariglio N, Haklai R, Rechavi G, Kloog Y. Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Israel. PURPOSE: Our aim was to examine whether active Ras and MycN cooperation contributes to the malignant phenotype of human neuroblastoma with amplified MycN gene, an aggressive incurable tumor. EXPERIMENTAL DESIGN: Human neuroblastoma LAN-1 cells, in which the MycN gene is amplified, were used to examine the impact of the Ras inhibitor farnesylthiosalicylic acid on cell growth, on the levels Ras and MycN proteins, and on profiles of gene expression. RESULTS: We show that LAN-1 cells express relatively large amounts of MycN and active Ras-GTP. Inhibition of active Ras by farnesylthiosalicylic acid led to attenuation of the Raf-MEK-ERK and phosphoinositide 3-kinase-Akt-glycogen synthase-3 (GSK-3) pathways, to reduction in cyclin D1, phospho-retinoblastoma, and E2F, and to increase in the cyclin-dependent kinase inhibitor p27 and in retinoblastoma-binding protein-1, an inhibitor of E2F transcriptional activity. Ras inhibition by farnesylthiosalicylic acid or by a dominant-negative Ras also led to complete disappearance of MycN protein from the nuclei of LAN-1 cells. This was a result of blocking of Akt inactivation of GSK-3, leading to GSK-3-dependent phosphorylation with consequent proteosomal degradation of MycN. Loss of active Ras and of MycN in LAN-1 cells was manifested in profiles of gene expression that could be expected from the loss of MycN transcriptional activity and of Ras signaling. These changes explain the farnesylthiosalicylic acid-induced inhibition of LAN-1 cell growth. CONCLUSIONS: Active Ras is needed to block MycN degradation, promoting cooperative Ras- and MycN-dependent cell cycle progression in LAN-1 cells. Ras inhibitors are therefore likely candidates for the treatment of advanced neuroblastoma characterized by high expression of MycN. PMID: 15958613 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Cancer Lett. 2005 Oct 18;228(1-2):21-7. MDM2 as MYCN transcriptional target: implications for neuroblastoma pathogenesis. Slack A, Lozano G, Shohet JM. Department of Pediatrics, Texas Children's Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. MYCN amplification is associated with an exceptionally poor prognosis in neuroblastoma. Furthermore, the crucial effectors of MYCN responsible for this aggressive subset of neuroblastoma await characterization. A critical negative regulator of the p53 tumor suppressor, MDM2, has been recently characterized in neuroblastoma cell lines as a transcriptional target of MYCN. Targeted inhibition of MYCN results in reduced MDM2 expression levels, with concomitant stabilization of p53 and stimulation of apoptosis in MYCN amplified neuroblastoma cell lines. These data suggest the possibility that MYCN-driven expression of MDM2 might play a role in counterbalancing the p53-dependent apoptotic pathways concurrently stimulated by over expression of MYC proteins. Mouse models of lymphoma have demonstrated that MDM2 expression, with decreased p53 activity, is critical for complete MYCC driven tumorigenesis. Our data suggest that a similar situation may apply for MYCN in neuroblastoma. Strategies for pharmacologic and genetic inhibition of MDM2 may prove to be an important new therapeutic approach in neuroblastoma. PMID: 15927364 [PubMed - in process] --------------------------------------------------------------- 9: Cancer Lett. 2005 Oct 18;228(1-2):97-104. Molecular mechanism of HMGA1 deregulation in human neuroblastoma. Giannini G, Cerignoli F, Mellone M, Massimi I, Ambrosi C, Rinaldi C, Gulino A. Department of Experimental Medicine and Pathology, Department of Pediatrics, University La Sapienza, Policlinico Umberto 1, Viale Regina Elena, 324, 00161 Rome, Italy. giuseppe.giannini@uniroma1.it Very soon after their original identification in HeLa cells in 1983, HMGA proteins appeared as interesting cancer-related molecules. Indeed, they were immediately noted as a sub-class of High Mobility Group proteins induced in fibroblast or epithelial cells transformed with sarcoma viruses. After more than 20 years, the association between HMGA protein expressions and cellular transformation has been largely confirmed and HMGA are among the most widely expressed cancer-associated proteins. Nevertheless, their functional contribution to tumour development and progression is far from being completely understood. Furthermore, although HMGA1 expression has been reported to be inducible by a number of factors and circumstances, the question of how their expression is deregulated in cancer is even less clear and somehow has been ignored from most researchers. An active AP1 site is the only characterized element of the HMGA1 human promoter, that remains a rather complicated and unexplored source of information to answer this question. Following the indication that c-Myc might bind and activate the mouse HMGA1 gene promoter, we have demonstrated that HMGA1 is a new target for MYCN in human neuroblastomas. In this report, we overview part of the current information on HMGA1 and focus our attention on the analysis of its human promoter. PMID: 15923078 [PubMed - in process] --------------------------------------------------------------- 10: Mol Cancer Ther. 2005 May;4(5):779-86. Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis. Tonelli R, Purgato S, Camerin C, Fronza R, Bologna F, Alboresi S, Franzoni M, Corradini R, Sforza S, Faccini A, Shohet JM, Marchelli R, Pession A. Unita di Terapia Cellulare, Dipartimento di Scienze Pediatriche Mediche e Chirurgiche, Policlinico Sant'Orsola-Malpighi, via Massarenti 11, 40138 Bologna, Italy. pession@med.unibo.it We developed an anti-gene peptide nucleic acid (PNA) for selective inhibition of MYCN transcription in neuroblastoma cells, targeted against a unique sequence in the antisense DNA strand of exon 2 of MYCN and linked at its NH(2) terminus to a nuclear localization signal peptide. Fluorescence microscopy showed specific nuclear delivery of the PNA in six human neuroblastoma cell lines: GI-LI-N and IMR-32 (MYCN-amplified/overexpressed); SJ-N-KP and NB-100 (MYCN-unamplified/low-expressed); and GI-CA-N and GI-ME-N (MYCN-unamplified/unexpressed). Antiproliferative effects were observable at 24 hours (GI-LI-N, 60%; IMR-32, 70%) and peaked at 72 hours (GI-LI-N, 80%; IMR-32, 90%; SK-N-KP, 60%; NB-100, 50%); no reduction was recorded for GI-CA-N and GI-ME-N (controls). In MYCN-amplified/overexpressed IMR-32 cells and MYCN-unamplified/low-expressed SJ-N-KP cells, inhibition was recorded of MYCN mRNA (by real-time PCR) and N-Myc (Western blotting); these inhibitory effects increased over 3 days after single treatment in IMR-32. Anti-gene PNA induced G(1)-phase accumulation (39-53%) in IMR-32 and apoptosis (56% annexin V-positive cells at 24 hours in IMR-32 and 22% annexin V-positive cells at 48 hours in SJ-N-KP). Selective activity of the PNA was shown by altering three point mutations, and by the observation that an anti-gene PNA targeted against the noncoding DNA strand did not exert any effect. These findings could encourage research into development of an anti-gene PNA-based tumor-specific agent for neuroblastoma (and other neoplasms) with MYCN expression. PMID: 15897242 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 11: Mol Cell Biol. 2005 Mar;25(6):2395-405. The novel ETS factor TEL2 cooperates with Myc in B lymphomagenesis. Cardone M, Kandilci A, Carella C, Nilsson JA, Brennan JA, Sirma S, Ozbek U, Boyd K, Cleveland JL, Grosveld GC. Department of Genetics, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, TN 38105, USA. The human ETS family gene TEL2/ETV7 is highly homologous to TEL1/ETV6, a frequent target of chromosome translocations in human leukemia and specific solid tumors. Here we report that TEL2 augments the proliferation and survival of normal mouse B cells and dramatically accelerates lymphoma development in Emu-Myc transgenic mice. Nonetheless, inactivation of the p53 pathway was a hallmark of all TEL2/Emu-Myc lymphomas, indicating that TEL2 expression alone is insufficient to bypass this apoptotic checkpoint. Although TEL2 is infrequently up-regulated in human sporadic Burkitt's lymphoma, analysis of pediatric B-cell acute lymphocytic leukemia (B-ALL) samples showed increased coexpression of TEL2 and MYC and/or MYCN in over one-third of B-ALL patients. Therefore, TEL2 and MYC also appear to cooperate in provoking a cadre of human B-cell malignancies. PMID: 15743832 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 12: J Clin Oncol. 2005 Feb 1;23(4):880-8. Relationship between MYCN copy number and expression in rhabdomyosarcomas and correlation with adverse prognosis in the alveolar subtype. Williamson D, Lu YJ, Gordon T, Sciot R, Kelsey A, Fisher C, Poremba C, Anderson J, Pritchard-Jones K, Shipley J. Molecular Cytogenetics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom. PURPOSE: Amplification of the transcription factor MYCN is an important molecular diagnostic tool in stratifying treatment for neuroblastoma. Increased copy number and overexpression of MYCN in the pediatric cancer rhabdomyosarcoma has been described in a number of small studies with conflicting conclusions about its association with clinicopathologic characteristics. We aimed to study the phenomenon in the largest series to date. PATIENTS AND METHODS: Using quantitative polymerase chain reaction, we measured MYCN copy number and expression levels in rhabdomyosarcoma samples from 113 and 92 individuals with a confirmed diagnosis of rhabdomyosarcoma, respectively. RESULTS: Increased copy number of MYCN was found to be a feature of both the embryonal and alveolar subtypes. The copy number and expression levels were significantly greater in the alveolar subtype, although the range of expression in both subtypes spanned several orders of magnitude. MYCN copy number showed a significant correlation with expression in the alveolar subtype; this relationship between copy number and expression could be modeled as a logarithmic function. It is notable that relatively high expression frequently occurred in embryonal rhabdomyosarcoma without high copy number and that low expression was found in some cases with high copy number. In patients with alveolar rhabdomyosarcoma, overexpression (greater than median) or gain of genomic copies of MYCN were significantly associated with adverse outcome. CONCLUSION: MYCN deregulation is a feature of rhabdomyosarcoma tumorigenesis, defines groups of patients with a poor prognosis, and is a potential target for novel therapies. PMID: 15681534 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 13: Proc Natl Acad Sci U S A. 2005 Jan 18;102(3):731-6. Epub 2005 Jan 11. The p53 regulatory gene MDM2 is a direct transcriptional target of MYCN in neuroblastoma. Slack A, Chen Z, Tonelli R, Pule M, Hunt L, Pession A, Shohet JM. Center for Cell and Gene Therapy, Texas Children's Cancer Center, Baylor College of Medicine, 1102 Bates Street, Houston, TX 77030, USA. The MYCN oncogene is the major negative prognostic marker in neuroblastoma with important roles in both the pathogenesis and clinical behavior of this aggressive malignancy. MYC oncogenes activate both proliferative and apoptotic cellular pathways and, accordingly, inhibition of p53-mediated apoptosis is a prerequisite for MYC-driven tumorigenesis. To identify novel transcriptional targets mediating the MYCN-dependent phenotype, we screened a MYCN-amplified neuroblastoma cell line by using chromatin immunoprecipitation (ChIP) cloning. We identified the essential p53 inhibitor and protooncogene MDM2 as a putative target. MDM2 has multiple p53-independent functions modulating cell cycle and transcriptional events. Standard ChIP with MYCN antibodies established the binding of MYCN to a consensus E-box within the human MDM2 promoter. Oligonucleotide pull-down assays further established the capacity of MYCN to bind to this promoter region, confirming the ChIP results. Luciferase reporter assays confirmed the E-box-specific, MYCN-dependent regulation of the MDM2 promoter in MYCN-inducible neuroblastoma cell lines. Real-time quantitative PCR and Western blot analysis demonstrated a rapid increase in endogenous MDM2 mRNA and MDM2 protein upon induction of MYCN. Targeted inhibition of MYCN in a MYCN-amplified neuroblastoma cell line resulted in decreased MDM2 expression levels with concomitant stabilization of p53 and induction of apoptosis. Our finding that MYCN directly modulates baseline MDM2 levels suggests a mechanism contributing to the pathogenesis of neuroblastoma and other MYC-driven malignancies through inhibition of MYC-stimulated apoptosis. PMID: 15644444 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 14: J Biol Chem. 2005 Mar 11;280(10):9474-81. Epub 2005 Jan 4. Linking of N-Myc to death receptor machinery in neuroblastoma cells. Cui H, Li T, Ding HF. Department of Biochemistry and Cancer Biology, Medical College of Ohio, Toledo, Ohio 43614, USA. The oncogene MYCN is amplified in aggressive neuroblastomas in which caspase-8, an essential component of death receptor pathways, is frequently inactivated, suggesting a critical role of death receptor-mediated apoptosis in suppression of N-Myc oncogenic activity. Elevated levels of N-Myc sensitize neuroblastoma cells to apoptosis induced by various death ligands. Using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis as a model, we define the mechanism underlying the sensitization effect. In neuroblastoma cells with increased expression of N-Myc, TRAIL triggers high levels of caspase-8 activation and Bid cleavage, leading to release of cytochrome c and Smac/DIABLO from mitochondria. However, the apoptotic process requires Smac/DIABLO, but not cytochrome c-mediated caspase-9 activation. N-Myc sensitizes neuroblastoma cells to TRAIL by up-regulating TRAIL receptor-2/DR5/KILLER and Bid. Moreover, DR5 mRNA is increased after N-Myc overexpression, and the human DR5 promoter contains two noncanonical E-boxes critical for the transcriptional activation by N-Myc. These findings establish a mechanistic link between N-Myc and death receptor machinery, which may serve as a checkpoint to guard the cell from N-Myc-initiated tumorigenesis. PMID: 15632181 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 15: Clin Cancer Res. 2004 Dec 15;10(24):8704-19. Induction of apoptosis by flavopiridol in human neuroblastoma cells is enhanced under hypoxia and associated with N-myc proto-oncogene down-regulation. Puppo M, Pastorino S, Melillo G, Pezzolo A, Varesio L, Bosco MC. Laboratory of Molecular Biology, Giannina Gaslini Institute, Genoa, Italy. PURPOSE: Neuroblastoma is the most common extracranial solid tumor of children that arises from the sympathetic nervous system. Survival rates for neuroblastoma patients is low despite intensive therapeutic intervention, and the identification of new effective drugs remains a primary goal. The cyclin-dependent kinase inhibitor, flavopiridol, has demonstrated growth-inhibitory and cytotoxic activity against various tumor types. Our aim was to investigate flavopiridol effects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of its activity by hypoxia, a critical determinant of tumor progression and a major challenge of therapy. EXPERIMENTAL DESIGN: Cell viability was monitored by 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazolium bromide (MTT) and trypan blue dye exclusion assays; DNA synthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa staining, DNA fragmentation, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling reaction, flow cytometric determination of hypodiploid DNA content, and evaluation of caspase activity and cytochrome c (CytC) release; MYCN expression was determined by Northern and Western blotting. RESULTS: Flavopiridol caused dose- and time-dependent decreases in neuroblastoma viability by inducing apoptosis, as confirmed by morphologic and biochemical criteria. Cell death was preceded by DNA synthesis inhibition and G1-G2 arrest, reversed by the pancaspase inhibitor, zVAD-fmk, and associated with caspase-3 and -2 activation and CytC increase. Moreover, flavopiridol strongly down-regulated MYCN mRNA and protein expression. Exposure to hypoxia enhanced both the extent of apoptosis and flavopiridol effects on CytC, caspase 3, and MYCN. CONCLUSIONS: These results indicate that flavopiridol has growth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease. PMID: 15623656 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 16: Oncogene. 2005 Feb 24;24(9):1533-41. Altered expression of cell cycle genes distinguishes aggressive neuroblastoma. Krasnoselsky AL, Whiteford CC, Wei JS, Bilke S, Westermann F, Chen QR, Khan J. Oncogenomics Section, Pediatric Oncology Branch, Advanced Technology Center, National Cancer Institute, 8717 Grovemont Circle, Gaithersburg, MD 20877, USA. In this study, gene expression profiling was performed on 103 neuroblastoma (NB) tumors, stages 1-4 with and without MYCN amplification, using cDNA microarrays containing 42,578 elements. Using principal component analysis (PCA) to analyse the relationships among these samples, we confirm that the global patterns of gene expression reflect the phenotype of the tumors. To explore the biological processes that may contribute to increasing aggressive phenotype of the tumors, we utilized a statistical approach based on PCA. We identified a specific subset of the cell cycle and/or chromosome segregation genes that distinguish stage 4 NB tumors from all lower stage tumors, including stage 3. Furthermore, the control of the kinetochore assembly emerges from the Gene Ontology analysis as one of the key biological processes associated with an aggressive NB phenotype. Finally, we establish that these genes are further upregulated in the most aggressive MYCN-amplified tumors. PMID: 15592497 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 17: Oncogene. 2005 Jan 20;24(4):680-7. High activin A-expression in human neuroblastoma: suppression of malignant potential and correlation with favourable clinical outcome. Schramm A, von Schuetz V, Christiansen H, Havers W, Papoutsi M, Wilting J, Schweigerer L. Abteilung fur Hamatologie, Onkologie und Endokrinologie, Universitats-Kinderklinik Essen, Germany. Amplification of the MYCN oncogene contributes to the malignant progression of human neuroblastomas, but the mechanisms have remained unclear. We have previously demonstrated that N-Myc facilitates angiogenesis by downregulating an angiogenesis inhibitor identified as the inhibin betaA homodimer activin A. Here, we have sought to define the molecular, biological and clinical consequences of activin A expression in human neuroblastoma. We report that enhanced activin A expression suppresses proliferation and colony formation of human neuroblastoma cells with amplified MYCN in vitro; that it inhibits neuroblastoma growth and angiogenesis in vivo; that it is highly expressed in differentiated, but not undifferentiated human neuroblastomas; and that it correlates with favourable outcome of neuroblastoma patients. Our results indicate that high activin A expression plays an important beneficial role in human neuroblastoma. PMID: 15580313 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 18: Eur J Cancer. 2004 Dec;40(18):2753-9. Comment in: Eur J Cancer. 2004 Dec;40(18):2639-42. MYCN-status in neuroblastoma: characteristics of tumours showing amplification, gain, and non-amplification. Spitz R, Hero B, Skowron M, Ernestus K, Berthold F. University of Cologne, Children's Hospital, Paediatric Oncology, Joseph-Stelzmann-Str. 9, Koln 50924, Germany. ruediger.spitz@medizin.uni-koeln.de While the role of MYCN-amplification (MNA) for risk assessment in neuroblastoma is undisputed, the phenomenon of gene copy excess below the amplification threshold is rarely described. To discuss biological characteristics and the clinical impact of the so-called MYCN-gain versus amplified or non-amplified cases, we investigated the MYCN status of 659 patients uniformly analysed by fluorescence in situ hybridisation. The number of MYCN-amplified tumours in our cohort was 18% (116/659); an additional 38 tumours (6%) displayed MYCN-gain. Both alterations were associated with an advanced stage disease, an increased patient age and further chromosomal alterations. Most of the amplified neuroblastomas displayed 1p aberrations, whereas MYCN-gain tumours correlated with 11q alterations. In contrast to the amplified cases, tumours with gain displayed no increased MYCN RNA levels. MNA versus non-amplification discriminated between good and poor outcomes, independent of stage, age and the degree of amplification. However, patients with amplified tumours showed a significantly better outcome when this was combined with non-stage 4 disease and age <1 year versus stage 4 and age < 1 year. Although MYCN-gain was associated with poor event-free-survival (EFS) in stages 1-3, 4S (P=0.005), this might be related to associated genetic aberrations and not to the MYCN-gain itself. A survival difference between neuroblastomas with gain and single copy MYCN could not be delineated. In conclusion, MNA predicts a poor outcome for neuroblastoma patients of all stages and age. MYCN-gain is also a characteristic feature of advanced stage tumours and older patients, but is not associated with higher MYCN expression and appears not to be discriminative in predicting patient outcome. PMID: 15571958 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 19: Eur J Cancer. 2004 Dec;40(18):2639-42. Comment on: Eur J Cancer. 2004 Dec;40(18):2753-9. MYCN amplification remains prognostically strong 20 years after its "clinical debut". Cohn SL, Tweddle DA. Publication Types: Comment Editorial PMID: 15571946 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 20: Proc Natl Acad Sci U S A. 2004 Nov 16;101(46):16251-6. Epub 2004 Nov 4. A screen for genes that suppress loss of contact inhibition: identification of ING4 as a candidate tumor suppressor gene in human cancer. Kim S, Chin K, Gray JW, Bishop JM. The G. W. Hooper Research Foundation and Department of Microbiology and Immunology, Department of Laboratory Medicine and Comprehensive Cancer Center, University of California, San Francisco, CA 94143, USA. suwon@itsa.ucsf.edu We have devised a screen for genes that suppress the loss of contact inhibition elicited by overexpression of the protooncogene MYCN. The initial application of this screen detected nine distinctive suppressors within a representative human cDNA library. One of these genes was ING4, a potential tumor suppressor gene that maps to human chromosome 12p13. Ectopic expression of ING4 suppressed the loss of contact inhibition elicited by either MYCN or MYC but had no direct effect on cellular proliferation. Pursuing the possibility that ING4 might be a tumor suppressor gene, we found inactivating mutations in ING4 transcripts from various human cancer cell lines. In addition, we used comparative genomic hybridization to detect deletion of the ING4 locus in 10-20% of human breast cancer cell lines and primary breast tumors. Ectopic expression of ING4 attenuated the growth of T47D human breast cancer cells in soft agar. We conclude that ING4 is a strong candidate as a tumor suppressor gene. PMID: 15528276 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 21: Cancer. 2004 Oct 15;101(8):1873-81. TrkA expression in peripheral neuroblastic tumors: prognostic significance and biological relevance. Shimada H, Nakagawa A, Peters J, Wang H, Wakamatsu PK, Lukens JN, Matthay KK, Siegel SE, Seeger RC. Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles and University of Southern California Keck School of Medicine, Los Angeles, California 90027, USA. hshimada@chla.usc.edu BACKGROUND: This study was conducted to investigate the prognostic significance and biologic relevance of trkA expression levels in peripheral neuroblastic tumors (pNTs) (i.e., neuroblastoma, ganglioneuroblastoma, and ganglioneuroma). METHODS: Levels of trkA expression from a total of 265 pNTs were determined by quantitative polymerase chain reaction analysis with Genescan software. The results were analyzed according to histopathology (favorable histology [FH] vs. unfavorable histology [UH] according to the International Neuroblastoma Pathology Classification) and MYCN tumor status (amplified vs. nonamplified) along with clinical stage and outcomes of the patients. RESULTS: The levels of trkA expression differed significantly between the group of patients who were alive and well (n = 170 patients) and the group that had progressed or died (n = 95 patients) and between the group that was alive (n = 188 patients) and the group that died (n = 77 patients). However, the trkA expression levels were not independent predictors of clinical outcome when the proportional hazards model contained the known prognostic variables of clinical stage, histopathology, and MYCN status (all tests were done in 196 patients). In the neuroblastoma category (n = 173 tumors), tumors in the FH/nonamplified MYCN subset (n = 112 tumors) expressed higher levels of trkA and showed an age-dependent neuroblastic differentiation: They were classified into either a poorly differentiated subtype (n = 91 tumors; all patients age < 1.5 years at diagnosis) or a differentiating subtype (n = 21 tumors; 57% of patients ages 1.5-5.0 years). Tumors in the UH/amplified MYCN subset (n = 30 tumors) expressed significantly lower levels of trkA and showed very limited neuroblastic differentiation. Tumors in the FH/amplified MYCN subset were very rare (n = 3 tumors) and expressed higher levels of trkA. Tumors in the UH/nonamplified MYCN subset (n = 28 tumors) had trkA levels in a wide range and showed limited neuroblastic differentiation. CONCLUSIONS: For patients with pNTs, levels of trkA expression did not add significant information to prognostic grouping, as defined by the combination of clinical stage, histopathology, and MYCN status. There was a biologically relevant correlation between molecular properties (trkA expression and MYCN status) and histopathologic features of the tumors in the neuroblastoma category. PMID: 15386308 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 22: Cancer. 2004 Sep 1;101(5):1081-9. High-dose chemotherapy followed by locoregional irradiation improves the outcome of patients with international neuroblastoma staging system Stage II and III neuroblastoma with MYCN amplification. Laprie A, Michon J, Hartmann O, Munzer C, Leclair MD, Coze C, Valteau-Couanet D, Plantaz D, Carrie C, Habrand JL, Bergeron C, Chastagner P, Defachelles AS, Delattre O, Combaret V, Benard J, Perel Y, Gandemer V, Rubie H; Neuroblastoma Study Group of the French Society of Pediatric Oncology. Departement d'Hemato-Oncologie, Hopital des Enfants, Toulouse, France. laprie@icr.fnclcc.fr BACKGROUND: The objective of this study was to determine whether systemic and regional, intensified treatment can improve the outcome of children who present with a localized neuroblastoma (NB) with MYCN amplification (MNA). METHODS: Between 1990 and 2000, 610 children with localized NB were included in the Localized Neuroblastoma 90 (NBL 90) and NBL 94 study from the French Society of Pediatric Oncology. Among them, 32 children had MNA with Stage II or III NB. During the first period of the study, 20 children (Group A) received postoperative conventional chemotherapy (CT) and/or radiotherapy (RT), depending on each patient's postoperative status. Subsequently, because of a high recurrence rate, the next 12 children (Group B) were given postoperative high-dose CT (HDC) (busulfan and melphalan) with stem cell rescue (SCR) followed by RT in addition to conventional postoperative CT. RESULTS: The two groups were comparable with regard to prognostic factors (age, location of the primary lesion, International Neuroblastoma Staging System stage, lymph node invasion) and response to preoperative CT. The 6-year overall survival rate was significantly different between the two groups 25% +/- 10% in Group A vs. 83% +/- 11% in Group B; P = 0.004). CONCLUSIONS: Postoperative intensification treatment with HDC, SCR, and locoregional RT resulted in higher survival rates when compared with standard treatment alone and should be considered in the treatment plan for children who are diagnosed with Stage II or III NB and MYCN amplification. Copyright 2004 American Cancer Society. Publication Types: Multicenter Study PMID: 15329919 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 23: Clin Cancer Res. 2004 Aug 15;10(16):5482-93. Combined histopathological and molecular cytogenetic stratification of medulloblastoma patients. Lamont JM, McManamy CS, Pearson AD, Clifford SC, Ellison DW. Northern Institute for Cancer Research, University of Newcastle, Newcastle-upon-Tyne, United Kingdom. This study examined the utility of stratifying children with medulloblastomas by a combination of refined histopathological classification and molecular cytogenetic evaluation. Detailed histopathological classification of tumors from a cohort of patients (n = 87) composed mainly of children entered into the International Society of Pediatric Oncology (SIOP)/United Kingdom Children's Cancer Study Group PNET3 trial (n = 65), included identification of the large cell/anaplastic phenotype. Fluorescence in situ hybridization was used to detect chromosome 17 abnormalities, losses of 9q22 and 10q24, and amplification of the MYCC and MYCN oncogenes. The large cell/anaplastic phenotype, which was present in 20% of medulloblastomas, emerged as an independent prognostic indicator. Loss of 17p13.3 (38% of medulloblastomas) was found across all of the histopathological variants, whereas MYCC/MYCN amplification (6%/8% of medulloblastomas) was significantly associated with the large cell/anaplastic phenotype. Both of these genetic abnormalities emerged as prognostic indicators. Loss of 9q22 was associated with the nodular/desmoplastic medulloblastoma variant, whereas loss of 10q24 was found in all of the variants. Together with metastatic tumor at presentation, the large cell/anaplastic phenotype, 17p13.3 loss, or high-frequency MYC amplification defined a high-risk group of children whose outcome was significantly (P = 0.0002) poorer than a low-risk group without these tumor characteristics. Combined evaluation of novel histopathological features and molecular cytogenetic abnormalities promises to allow stratification of patients with medulloblastoma, such that those likely to be cured will be spared the side effects of maximal therapy, which can be targeted at those with aggressive disease. PMID: 15328187 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 24: Cancer Genet Cytogenet. 2004 Aug;153(1):10-5. Alternative pathways of MYCN gene copy number increase in primary neuroblastoma tumors. Valent A, Guillaud-Bataille M, Farra C, Lozach F, Spengler B, Terrier-Lacombe MJ, Valteau-Couanet D, Danglot G, Lenoir GM, Brison O, Benard J, Bernheim A. Laboratoire de Genomique Cellulaire des Cancers, Institut Gustave Roussy, Unite Mixte de Recherche 8125, Centre National de la Recherche Scientifique, rue Camille Desmoulins 39, 94805 Villejuif Cedex, France. avalent@igr.fr Neuroblastomas, tumors of the sympathetic nervous system, account for 7-10% of the cancers of childhood. Genetic studies have shown, and this study has confirmed, that neuroblastomas are very heterogeneous; no single genetic change common to all neuroblastomas has yet been identified. One genetic aberration found frequently in this pediatric tumor is MYCN gene amplification. Recently we identified a new subset of tumors showing MYCN gain (small increases in gene number arising from unbalanced translocation). To investigate whether gain precedes amplification or is an independent event, we surveyed 200 primary tumors for MYCN copy number with fluorescence in situ hybridization; 152 of 200 (76%) were MYCN single-copy tumors, whereas 48 of 200 (24%) tumors harbored MYCN abnormalities: 36 of the 48 (75%) had MYCN amplification and 12 (25%) had MYCN gain. Among the 36 with MYCN amplified gene, we found four that also showed gain. In three tumors exhibiting simultaneous gain and amplification, these two events were detected in neighboring cells. In the fourth case we detected only MYCN gain in metastatic neuroblasts in the bone marrow, but both MYCN amplification and gain in the primary tumor. The detailed study of these four cases suggests that there may be several different mechanisms leading to increase in MYCN copy number. Further studies in other human malignancies are necessary to determine whether simultaneous gain and amplification are specific to neuroblastoma or constitute a general mechanism by which tumor cells can acquire selective growth advantage. PMID: 15325088 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 25: J Pediatr Hematol Oncol. 2004 Aug;26(8):484-7. Eukaryotic initiation factor 4E staining as a clinical marker in pediatric neuroblastoma. Parker A, Anderson C, Weiss KL, Grimley M, Sorrells D. Department of Pediatrics, San Antonio Military Pediatrics Center, San Antonio, Texas 78236-5300, USA. amy.parker@lackland.af.mil OBJECTIVES: Neuroblastoma (NBL) has several well-established prognostic factors. Eukaryotic initiation factor 4E (4E) has shown promise as a prognostic marker in other cancers. We hypothesize that a correlation exists between 4E staining and NBL clinical outcome. METHODS: Eleven adrenal NBL patient charts were reviewed for data, including age, stage, MYCN amplification, Shimada classification, and mortality. These patients' surgical specimens were stained with 4E antibody and scored for staining density. 4E expression, quantified by staining density, was compared to clinical data. CONCLUSION: 4E staining significantly correlates with age at diagnosis. The remaining prognostic factors lack statistically significant correlation. Increasing sample size may further establish statistically significant correlations. 4E could become an additional prognostic factor of NBL. PMID: 15284584 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 26: J Cell Biochem. 2004 Aug 15;92(6):1282-95. Expression and DNA-binding activity of MYCN/Max and Mnt/Max during induced differentiation of human neuroblastoma cells. Smith AG, Popov N, Imreh M, Axelson H, Henriksson M. Department of Laboratory Medicine, Molecular Medicine, Lund University, University Hospital MAS, S-205 02 Malmo, Sweden. Amplification of MYCN is one of the most important prognostic markers for neuroblastoma and is correlated with rapid tumor progression and poor prognosis. MYCN belongs to the Myc/Max/Mad/Mnt network of proteins that regulate proliferation, apoptosis, and differentiation. It is well established that MYCN is downregulated during induced differentiation of neuroblastoma cells carrying an amplified MYCN gene, but very little is known about other components of the network, i.e., the Max, Mad, and Mnt proteins, during this process. In this study we show that Mad and Mnt expression was only modestly regulated in differentiating SK-N-BE(2) neuroblastoma cells, while MYCN was rapidly downregulated. This downregulation was reflected in a decreased MYCN/Max DNA-binding activity while the Mnt/Max binding did not change during differentiation. In parallel experiments we also analyzed the Myc/Max/Mad expression and DNA binding capacity during induced differentiation in the MYCN single copy neuroblastoma cell line SH-SY5Y. In this cell line only modest changes in expression of the components of the MYCN/Max/Mad/Mnt network was detected, but since the cell line expresses relatively low levels of MYCN and c-Myc, these changes might be of functional significance. Cell cycle analyses of SK-N-BE(2) demonstrated an increase in the G1-phase fraction after RA-treatment. These data show that the decreased MYCN expression and MYCN DNA-binding is correlated with retarded cell cycle progression. Furthermore, when Mad1 or Mnt was overexpressed in SK-N-BE(2) cells they retained the capacity to differentiate, underscoring the notion that MYCN downregulation, and not changes in Mad/Mnt expression, is essential for neuroblastoma cell differentiation. Copyright 2004 Wiley-Liss, Inc. PMID: 15258910 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 27: Clin Cancer Res. 2004 Jul 1;10(13):4307-13. The tumor-associated antigen PRAME is universally expressed in high-stage neuroblastoma and associated with poor outcome. Oberthuer A, Hero B, Spitz R, Berthold F, Fischer M. Children's Hospital, Department of Pediatric Oncology and Hematology, University of Cologne, Cologne, Germany. andre.oberthuer@medizin.uni-koeln.de PURPOSE: The tumor-associated antigen PRAME, a potential candidate for immunotherapeutic targeting, is frequently expressed in a variety of cancers. However, no information about its presence in neuroblastoma is available to date. We therefore evaluated and quantified PRAME expression in a considerable number of neuroblastoma tumors and assessed its impact on the outcome of patients. EXPERIMENTAL DESIGN: Qualitative analysis of PRAME expression was assessed by reverse transcription (RT)-PCR screening of 94 patients with primary neuroblastoma. The same cohort was used for semiquantitative determination of transcript levels by Northern blotting, comparing the signal intensities of patients with those of testis total RNA. For more precise quantification of PRAME expression, real-time RT-PCR was performed in 88 patients of the above cohort and 7 additional patients, thus leaving a total of 101 patients that were analyzed with either method. Furthermore, association with tumor stage, age of patients at diagnosis, and MYCN amplification was determined as well as the prognostic impact of PRAME expression. RESULTS: RT-PCR screening detected PRAME expression in 93% of primary neuroblastoma and 100% of patients with advanced disease. Furthermore, RT-PCR and Northern blot analysis showed a highly significant association of PRAME expression with both higher tumor stage (P < 0.01) and the age of patients at diagnosis (P < 0.01). Finally, precise quantification of PRAME expression by quantitative real-time reverse transcription-PCR displayed significant impact on the outcome of patients. CONCLUSIONS: PRAME expression in neuroblastoma is extraordinarily common and was universally seen in patients with advanced-stage disease in our study. Furthermore, significant impact of PRAME expression on the outcome of patients was shown. Thus, PRAME may present a particularly attractive target for immunotherapeutic strategies in neuroblastoma. PMID: 15240516 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 28: Cytogenet Genome Res. 2004;106(1):49-54. Evolution of unbalanced gain of distal chromosome 2p in neuroblastoma. Stallings RL, Carty P, McArdle L, Mullarkey M, McDermott M, O'Meara A, Ryan E, Catchpoole D, Breatnach F. National Centre for Medical Genetics, Our Lady's Hospital for Sick Children, Crumlin, Dublin, Ireland. ray.stallings@olhsc.ie Neuroblastoma, one of the most common tumors of childhood, presents at diagnosis with a vast number of recurrent chromosomal imbalances that include hyperdiploidy for whole chromosomes, partial loss of 1p, 3p, 4p, 11q, 14q, partial gain of 1q, 7q, 17q and amplification of MYCN. These abnormalities are nonrandomly distributed in neuroblastoma as loss of 3p and 11q rarely occur in MYCN amplified neuroblastomas. Here, we report on a patient who had a non-MYCN amplified 3p-/11q- neuroblastoma at diagnosis who subsequently developed a high level of MYCN amplification in bone marrow metastases 41 months after induction of complete remission. The tumor at diagnosis had low level unbalanced gain of distal 2p. In order to assess the frequency of low level gain of distal 2p in neuroblastoma, we examined the comparative genomic hybridization results from 60 neuroblastomas. Among non-MYCN amplified neuroblastomas, 8/45 (18%) had low level gain of distal 2p. Low level gain for a segment of 2p (i.e. a region larger than the 2p23-->p24 undergoing amplification) was also detected in five of the 15 tumors that had high level MYCN amplification. The possibility that low level gain of distal 2p is a risk factor for high level MYCN amplification is discussed. Copyright 2004 S. Karger AG, Basel Publication Types: Case Reports PMID: 15218241 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 29: Int J Oncol. 2004 Jun;24(6):1457-66. Identification of novel human neuronal leucine-rich repeat (hNLRR) family genes and inverse association of expression of Nbla10449/hNLRR-1 and Nbla10677/hNLRR-3 with the prognosis of primary neuroblastomas. Hamano S, Ohira M, Isogai E, Nakada K, Nakagawara A. Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717, Japan. To search for novel prognostic indicators, we previously cloned >2,000 novel genes from primary neuroblastoma (NBL) cDNA libraries and screened for differential expression between the subsets with favorable (stage 1 or 2 with a single copy of MYCN) and unfavorable (stage 3 or 4 with amplification of MYCN) prognosis. From them, we have identified 3 genes of human neuronal leucine-rich repeat protein (NLRR) family: Nbla10449/hNLRR-1, Nbla00061/hNLRR-2/GAC1 and Nbla10677/hNLRR-3. An additional family member, hNLRR-5, was also found by homology search against public database. NLRR family proteins have been proposed to function as a neuronal adhesion molecule or soluble ligand binding receptor like Drosophila toll and slit with multiple domains including 11 sets of extracellular leucine-rich repeat (LRR)-motifs. However, the functional role of the NLRR protein family has been elusive. Our present study shows that hNLRR mRNAs are preferentially expressed in nervous system and/or adrenal gland. In cancer cell lines, hNLRR-1, hNLRR-3 and hNLRR-5 are expressed at high levels in the neural crest-derived cells. Most remarkably, in primary NBLs, hNLRR-1 is significantly expressed at high levels in unfavorable subsets as compared to favorable ones, whereas the expression pattern of hNLRR-3 and hNLRR-5 is the opposite. In order to understand the function of these receptors, we have used newborn mouse superior cervical ganglion (SCG) cells which are dependent on nerve growth factor (NGF) for their survival. Expression of the mouse counterparts of hNLRR-2 and hNLRR-3 is up-regulated after NGF-induced differentiation and down-regulated after NGF depletion-induced apoptosis. On the other hand, expression of hNLRR-1 and hNLRR-5 is inversely regulated in the same system. These results have suggested that the regulation of the hNLRR family genes may be associated with NGF signaling pathway in both SCG cells and neuroblastoma. Our quantitative real-time RT-PCR analysis using 99 primary NBLs has revealed that high levels of hNLRR-1 expression are significantly associated with older age (>1 year, p=0.0001), advanced stages (p=0.0007), low expression of TrkA (p=0.011), and MYCN amplification (p=0.0001), while those of hNLRR-3 expression are significantly correlated with the favorable prognostic indicators. Furthermore, multivariate analysis reveals that expression of hNLRR-1 is an independent prognostic indicator in human neuroblastoma. Thus, our results demonstrate that, despite being members of the same family, hNLRR-1 and hNLRR-3 may share different biological function among the NBL subsets, and that their expression level becomes novel prognostic indicators of NBL. PMID: 15138588 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 30: J Exp Med. 2004 May 3;199(9):1213-21. Natural killer T cells infiltrate neuroblastomas expressing the chemokine CCL2. Metelitsa LS, Wu HW, Wang H, Yang Y, Warsi Z, Asgharzadeh S, Groshen S, Wilson SB, Seeger RC. Department of Pediatrics, Division of Hematology-Oncology, MS #57, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles, CA 90027, USA. CD1d-restricted Valpha24-Jalpha18-invariant natural killer T cells (iNKTs) are potentially important in tumor immunity. However, little is known about their localization to tumors. We analyzed 98 untreated primary neuroblastomas from patients with metastatic disease (stage 4) for tumor-infiltrating iNKTs using TaqMan((R)) reverse transcription polymerase chain reaction and immunofluorescent microscopy. 52 tumors (53%) contained iNKTs, and oligonucleotide microarray analysis of the iNKT(+) and iNKT(-) tumors revealed that the former expressed higher levels of CCL2/MCP-1, CXCL12/SDF-1, CCL5/RANTES, and CCL21/SLC. Eight tested neuroblastoma cell lines secreted a range of CCL2 (0-21.6 ng/ml), little CXCL12 (50%) in repeated screens. This cell-based, MYCN reporter gene assay will be scaled up for high throughput screens of compound libraries and will aid in the future development of specific therapeutic strategies in neuroblastoma and other tumours in which MYCN amplification has been implicated. Publication Types: Review Review, Multicase PMID: 12880971 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 39: Cancer Lett. 2003 Jul 18;197(1-2):93-8. The p53 pathway and its inactivation in neuroblastoma. Tweddle DA, Pearson AD, Haber M, Norris MD, Xue C, Flemming C, Lunec J. Cancer Research Unit, The Medical School, University of Newcastle, Newcastle-upon-Tyne NE2 4HH, UK. d.a.tweddle@newcastle.ac.uk Early studies of p53 in neuroblastoma reported infrequent mutations in tumours and cell lines. Cytoplasmic sequestration was later proposed as an alternative mechanism of inactivation, but many studies have since reported an intact p53 pathway in neuroblastoma cell lines, as detected by nuclear p53 accumulation after DNA damage, intact DNA binding, transcriptional activation of target genes and the induction of apoptosis. In some MYCN amplified cell lines however, an irradiation induced G(1) arrest does not occur, despite the presence of normal p53. Neuroblastoma usually responds to chemotherapy but frequently relapses, and there is evidence from tumours, and cell lines that p53 inactivation via mutation or MDM2 amplification occurs at relapse and is sometimes associated with multidrug resistance. If p53 inactivation occurs frequently in relapsed tumours it may be appropriate to include p53 independent therapies in the initial management of high-risk neuroblastoma. Publication Types: Review Review, Tutorial PMID: 12880966 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 40: Cancer Lett. 2003 Jul 18;197(1-2):81-6. Genes co-amplified with MYCN in neuroblastoma: silent passengers or co-determinants of phenotype? Scott D, Elsden J, Pearson A, Lunec J. Cancer Research Unit, University of Newcastle Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH UK. Amplification of the MYCN oncogene in neuroblastoma is associated with poor prognosis. The amplified unit of DNA can be up to 1 Mb in size and so could contain additional genes that affect tumour phenotype. Identification of such genes may assist in optimising the determination of prognosis, and could provide new targets for treatment. Three genes have so far been identified, which are frequently co-amplified with MYCN in neuroblastoma, DDX1, NAG and N-cym. In this review, the known or putative properties of the protein products of the genes are discussed, and their possible roles in determining tumour behaviour are assessed. Publication Types: Review Review, Tutorial PMID: 12880964 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 41: Cancer Lett. 2003 Jul 18;197(1-2):11-7. Long-term results and risk profiles of patients in five consecutive trials (1979-1997) with stage 4 neuroblastoma over 1 year of age. Berthold F, Hero B, Kremens B, Handgretinger R, Henze G, Schilling FH, Schrappe M, Simon T, Spix C. Department of Pediatric Oncology and Hematology, Children's Hospital, University of Cologne, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany. frank.berthold@medizin.uni-koeln.de During the last two decades new diagnostic and therapeutic tools have been utilized to improve the poor survival chances of children with stage 4 neuroblastoma. This study reviews the risk profiles and the long-term outcome of patients from five consecutive German neuroblastoma trials. A total of 96% of all German patients registered at the German childhood cancer registry with neuroblastoma stage 4 over 1 year of age at diagnosis entered one of the trials during 1979-2001. Eight hundred and twenty-eight consecutive children were analyzed retrospectively. In spite of having significantly improved diagnostic tools like bone marrow superstaging and mIBG scintigraphy the stage 4 incidence did not increase after reaching completeness of the registry (5.4 cases/100,000 children at 1-14 years of age; P=0.52). The distribution of the primary tumors and of metastases was constant over the periods. The amount of bone marrow infiltration did not change with time. The risk factors lactate dehydrogenase, ferritin and MYCN, and the clinical risk groups 4A, 4B, 4C also remained constant over the trials with a few exceptions for NB97. The 5-year event free survival increased from 0.01+/-0.01 (NB79) to 0.14+/-0.03 (NB85), 0.16+/-0.04 (NB82), 0.27+/-0.02 (NB90), and 0.33+/-0.04 (NB97). The overall survival rates improved similarly from 0.04 (NB79) to 0.44 (NB97). In conclusion, the improved survival was associated with better treatment and not caused by lower risk profiles in stage 4 neuroblastoma patients. Publication Types: Meta-Analysis PMID: 12880954 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 42: Cancer Genet Cytogenet. 2003 Jul 15;144(2):125-33. Cytogenetic and molecular findings related to rhabdomyosarcoma. An analysis of seven cases. Gil-Benso R, Lopez-Gines C, Carda C, Lopez-Guerrero JA, Ferrer J, Pellin-Perez A, Llombart-Bosch A. Department of Pathology, Medical School, University of Valencia, Avda. Blasco Ibanez 17, Valencia 46010, Spain. Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in childhood. Histologically, it is subdivided histologically into two main subtypes: alveolar (ARMS) and embryonal (ERMS). ARMS is characterized by t(2;13)(q35;q14) or its variant t(1;13)(p36;q14), which fuse PAX3 and PAX7, respectively, with FKHR to produce chimeric genes. ERMS is frequently associated with loss of heterozygosity of 11p15.5. We investigated seven RMS (three ARMS and four ERMS) by means of cytogenetic, fluorescence in situ hybridization, and molecular analyses, including the study of the main genes implicated in the G1- to S-phase cell cycle transition, and correlated these studies with pathologic findings and clinical outcome. All tumors showed clonal, numerical, and structural chromosomal abnormalities. Two ARMS had the t(2;13)(q35;q14) and the third a PAX7/FKHR fusion, a cryptic t(1;13)(p36;q14), undetected by cytogenetic techniques, but revealed by reverse transcriptase polymerase chain reaction. One ERMS showed a der(11)t(3;11)(p21;p15) as a sole structural anomaly. Gene amplification was seen in four tumors, as double minutes or in the form of homogeneously staining regions. Overexpression of MYCN oncogene was found in two ARMS; N-myc DNA probe detected oncogene amplification located on the double minutes of these cases. Analysis of the regulatory genes responsible for G1- to S-phase transition showed a homozygous deletion of the 9p21 locus genes in a spindle-cell ERMS. PMID: 12850375 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 43: Eur J Cancer. 2003 Jul;39(10):1478-85. Aberrant methylation of multiple genes in neuroblastic tumours. relationship with MYCN amplification and allelic status at 1p. Gonzalez-Gomez P, Bello MJ, Lomas J, Arjona D, Alonso ME, Aminoso C, Lopez-Marin I, Anselmo NP, Sarasa JL, Gutierrez M, Casartelli C, Rey JA. Laboratorio de Oncogenetica Molecular, Dept. C. Experimental, Hospital Universitario La Paz, Paseo de la Castellana 261, 28046, Madrid Spain. Aberrant hypermethylation occurs in tumour cell CpG islands and is an important pathway for the repression of gene transcription in cancers. We investigated aberrant hypermethylation of 11 genes by methylation-specific polymerase chain reaction (PCR), after treatment of the DNA with bisulphite, and correlated the findings with MYCN amplification and allelic status at 1p in a series of 44 neuroblastic tumours. This tumour series includes five ganglioneuromas (G), one ganglioneuroblastoma (GN) and 38 neuroblastomas (six stage 1 tumours; five stage 2 tumours; six stage 3 cases; 19 stage 4 tumours, and two stage 4S cases). Aberrant methylation of at least one of the 11 genes studied was detected in 95% (42 of 44) of the cases. The frequencies of aberrant methylation were: 64% for thrombospondin-1 (THBS1); 30% for tissue inhibitor of metalloproteinase 3 (TIMP-3); 27% for O6-methylguanine-DNA methyltransferase (MGMT); 25% for p73; 18% for RB1; 14% for death-associated protein kinase (DAPK), p14ARF, p16INK4a and caspase 8, and 0% for TP53 and glutathione S-transferase P1 (GSTP1). No aberrant methylation was observed in four control normal tissue samples (brain and adrenal medulla). MYCN amplification was found in 11 cases (all stage 4 neuroblastomas), whereas allelic loss at 1p was identified in 16 samples (13 stage 4 and two stage 3 neuroblastomas, and one ganglioneuroma). All but one case with caspase 8 methylation also displayed MYCN amplification. Our results suggest that promoter hypermethylation is a frequent epigenetic event in the tumorigenesis of neuroblastic tumours, but no specific pattern of hypermethylated genes could be demonstrated. PMID: 12826052 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 44: Lab Invest. 2003 Jun;83(6):813-23. Expression of trkB in human neuroblastoma in relation to MYCN expression and retinoic acid treatment. Edsjo A, Lavenius E, Nilsson H, Hoehner JC, Simonsson P, Culp LA, Martinsson T, Larsson C, Pahlman S. Department of Laboratory Medicine, Lund University, University Hospital MAS, Malmo, Sweden. Expression of full-length trkB can be found in some highly malignant neuroblastoma tumors with an amplified MYCN gene. This contrasts sympathetic neuroblasts, from which neuroblastomas are thought to arise, which neither express trkB nor are dependent on the p145(trkB) ligands, brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5, for their normal development. In this study we show that trkB was expressed in two out of five neuroblastoma tumors with amplified MYCN, while no trkB expression was observed when the MYCN gene was overexpressed in a non-MYCN-amplified neuroblastoma cell line. This shows that MYCN overexpression per se is not sufficient to induce trkB expression. trkB expression and BDNF responsiveness in neuroblastoma cells can be induced by all-trans-retinoic acid (RA). When SH-SY5Y cells were stimulated with a combination of RA and BDNF, norepinephrine and tyrosine hydroxylase levels were unaltered, showing that the cells did not change toward a more catecholaminergic sympathetic phenotype. However, expression of growth-associated protein 43, indicative of a neuronal phenotype, was elevated. Vesicular acetylcholine transporter, choline acetyl transferase, and neuropeptide tyrosine mRNA levels also increased in RA-BDNF-treated cells, which could suggest that these cells develop into a sympathetic cholinergic phenotype. In addition, treatment with RA-induced expression of the platelet-derived growth factor receptor-alpha. As previously shown for BDNF, platelet-derived growth factor stimulated growth of the RA-treated cells, findings that could have clinical relevance. If these receptors mediate a mitogenic signal in vivo also, this might limit the effect of RA treatment on neuroblastoma patients. PMID: 12808116 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 45: Int J Cancer. 2003 Aug 20;106(2):147-52. c-Kit is preferentially expressed in MYCN-amplified neuroblastoma and its effect on cell proliferation is inhibited in vitro by STI-571. Vitali R, Cesi V, Nicotra MR, McDowell HP, Donfrancesco A, Mannarino O, Natali PG, Raschella G, Dominici C. Bambino Gesu Children's Hospital, Laboratory of Oncology, Rome, Italy. Coexpression for c-Kit receptor and its ligand stem cell factor (SCF) has been described in neuroblastoma (NB) cell lines and tumors, suggesting the existence of an autocrine loop modulating tumor growth. We evaluated c-Kit and SCF expression by immunohistochemistry in a series of 75 primary newly diagnosed neuroblastic tumors. Immunostaining for c-Kit was found in 10/75 and for SCF in 17/75, with 5/10 c-Kit-positive tumors also expressing SCF. For both, c-Kit and SCF staining were predominantly found in the most aggressive subset of tumors, i.e., those amplified for MYCN: c-Kit was detected in 8/14 amplified vs. 2/61 single copy (p<0.001), and SCF in 9/14 amplified vs. 8/61 single copy tumors (p<0.001). Furthermore, the association of c-Kit expression with advanced stage (3 or 4) (p=0.001) and of SCF expression with adrenal primary (p=0.03) was substantiated. The in vitro activity of the tyrosine kinase inhibitor STI-571 (imatinib mesylate, Gleevec, Glivec) on NB cell lines positive or negative for c-Kit was also assessed. When cells were grown in 10% fetal calf serum, the 4 c-Kit-positive cell lines tested were sensitive to STI-571 growth inhibition to a different extent (ranging from 30 to 80%); also the c-Kit-negative cell line GI-CA-N was slightly affected, suggesting that other STI-571 targets operate in regulating NB proliferation. In addition, c-Kit-positive cell lines SK-N-BE2(c) and HTLA230, grown in SCF only, remained sensitive (40 and 70% of growth inhibition, respectively), while, in the same conditions, proliferation of the c-Kit-negative cell line GI-CA-N was not affected. Immunoprecipitation of c-Kit from cell lysates of SK-N-BE2(c) and HTLA230 cells grown in SCF and subsequent western blot analysis of the immunoprecipitates revealed a sharp decrease of c-Kit phosphorylation after STI-571 treatment. These data demonstrate that both c-Kit and SCF are preferentially expressed in vivo in the most aggressive neuroblastic tumors and that their signaling is active in promoting in vitro NB cell proliferation that can be selectively inhibited by treatment with STI-571. Copyright 2003 Wiley-Liss, Inc. PMID: 12800187 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 46: Int J Oncol. 2003 Jun;22(6):1369-74. MGC9753 gene, located within PPP1R1B-STARD3-ERBB2-GRB7 amplicon on human chromosome 17q12, encodes the seven-transmembrane receptor with extracellular six-cystein domain. Katoh M, Katoh M. M&M Medical BioInformatics, Narashino 275-0022, Japan. mkatoh@ncc.go.jp MYC, ERBB2, MET, FGFR2, CCNE1, MYCN, WNT2, CD44, MDM2, NCOA3, IQGAP1 and STK6 loci are amplified in human gastric cancer. It has been reported that the gene corresponding to EST H16094 is co-amplified with ERBB2 gene in human gastric cancer. Here, we identified and characterized the gene corresponding to EST H16094 by using bioinformatics. BLAST programs revealed that EST H16094 was derived from the uncharacterized MGC9753 gene. Two ORFs were predicted within human MGC9753 mRNA, and ORF1 (nucleotide position 18-980 of NM_033419.1) was predicted as the coding region of human MGC9753 mRNA based on comparative genomics. Nucleotide sequence of mouse Mgc9753 mRNA was next determined in silico by modification of AK052486 cDNA (deleting C at the nucleotide position 37). Human MGC9753 and mouse Mgc9753 proteins were 320-amino-acid seven-transmembrane receptors with the N-terminal six-cysteine domain and an N-glycosylation site (85.0% total-amino-acid identity). Human MGC9753 protein showed 90.6% total-amino-acid identity with human CAB2 aberrant protein, which lacked the third-transmembrane domain of MGC9753 due to frame shifts within ORF. Human MGC9753 gene, consisting of eight exons, were clustered with PPP1R1B, STARD3, TCAP, PNMT, ERBB2, MGC14832 and GRB7 genes within the 120-kb region. PPP1R1B, STARD3, MGC9753, ERBB2 and GRB7 genes are co-amplified in several cases of gastric cancer. This is the first report on comprehensive characterization of the amplicon around the PPP1R1B-STARD3-TCAP-PNMT-MGC9753-ERBB2-MGC14832-GRB7 locus on human chromosome 17q12. PMID: 12739007 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 47: Virchows Arch. 2003 Jun;442(6):555-62. Epub 2003 Apr 23. Neuroblastic tumors associated with opsoclonus-myoclonus syndrome: histological, immunohistochemical and molecular features of 15 Italian cases. Gambini C, Conte M, Bernini G, Angelini P, Pession A, Paolucci P, Donfrancesco A, Veneselli E, Mazzocco K, Tonini GP, Raffaghello L, Dominici C, Morando A, Negri F, Favre A, De Bernardi B, Pistoia V. Service of Pathology, Giannina Gaslini Children's Hospital, Largo Gerolamo Gaslini 5, 16148, Genova, Italy. claudiogambini@ospedale-gaslini.ge.it The aim of this study was to investigate the histological, immunohistochemical and molecular features of a series of children with neuroblastic tumors (NTs) and opsoclonus-myoclonus syndrome (OMS). Of 1187 children (age 0-15 years) with previously untreated NTs registered between 1979 and 1995, 15 (1.3%) had OMS at presentation. The majority of patients showed favorable biological characteristics, such as lack of amplification of the neuroblastoma-associated avian myelocytomatosis homolog MYCN oncogene and aneuploid nuclear DNA content. Tumor histology was reviewed according to the International Neuroblastoma Pathology Classification. Histology of the 15 cases of NTs with OMS was ganglioneuroblastoma, intermixed, in 10 patients; ganglioneuroma, maturing, in 1; and neuroblastoma in 4. Of 15 tumors, 12 (10 ganglioneuroblastomas, 2 neuroblastomas) showed abundant interstitial or perivascular lymphoid infiltrates, the latter often organized in secondary lymphoid follicles. The three remaining cases had only minimal infiltrates. A review of 91 cases of age- and stage-matched neuroblastic tumors not associated with OMS tested as controls showed that the degree of lymphoid infiltration was significantly lower than that detected in OMS-related tumors. Furthermore, lymphoid follicles were always present in the latter tumors, whereas they were detected only in a few ganglioneuroma, intermixed tumors from the control group. In conclusion, ganglioneuroblastoma, intermixed subtype, lack of MYCN amplification, aneuploid DNA content and presence of lymphoid infiltrates may contribute to favorable prognosis in NTs associated with OMS. Publication Types: Multicenter Study PMID: 12709798 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 48: Cancer Lett. 2003 Apr 25;193(2):171-6. Yaf2 inhibits Myc biological function. Madge B, Geisen C, Moroy T, Schwab M. Division of Cytogenetics-H0400, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. The proto-oncogenes of the myelocytomatosis viral oncogene homolog (MYC) family, including MYC, MYCN and MYCL, encode nuclear proteins that act as transcription factors. The Myc protein is the best studied member of this family and is involved in cell cycle regulation, differentiation and cell death. We have previously demonstrated that the zinc-finger protein Yaf2 interacts with the central region of MycN and enhances MycN dependent transcriptional activation. Here we show that Yaf2 also binds to the Myc protein in vivo and in vitro. In contrast to the activating effect on MycN function, Yaf2 inhibits Myc mediated transactivation and transformation. This differential influence on two members of the Myc family gives insight into a new mechanism to modulate the biological activities of Myc transcription factors. PMID: 12706874 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 49: Cancer Res. 2003 Apr 1;63(7):1631-5. ID2 expression is not associated with MYCN amplification or expression in human neuroblastomas. Wang Q, Hii G, Shusterman S, Mosse Y, Winter CL, Guo C, Zhao H, Rappaport E, Hogarty MD, Maris JM. Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA. MYCN is a biologically and clinically important oncogene in human neuroblastoma as genomic amplification reliably predicts for aggressive tumor behavior and a poor prognosis. However, the mechanism by which MYCN amplification and overexpression contributes to a highly malignant phenotype remains obscure. ID2 is a dominant inhibitor of the RB1 tumor suppressor gene product and recently was suggested to be a direct transcriptional target of MYCN. Overexpression of Id2 protein has thus been postulated to result in functional inactivation of retinoblastoma in MYCN-amplified neuroblastomas, offering a potential explanation for the undifferentiated and highly proliferative nature of most MYCN-amplified neuroblastomas, as well as the paucity of retinoblastoma pathway mutations observed in clinical samples. We therefore sought to determine the likelihood that ID2 overexpression is associated with MYCN amplification and overexpression in human neuroblastoma. ID2 was not differentially expressed in 39 primary neuroblastoma specimens analyzed by oligonucleotide array-based expression analysis, and there was no correlation with MYCN expression levels. ID2 mRNA and protein expression was highly variable and independent of MYCN amplification status and mRNA expression in 10 human-derived neuroblastoma cell lines. In addition, ID2 mRNA expression was not associated with MYCN gene amplification status (P = 0.15) or MYCN expression (r = 0.22) in 131 separate diagnostic primary neuroblastoma samples analyzed by real-time quantitative RT-PCR. These data suggest that transcriptional regulation of ID2 by the MycN oncoprotein is unlikely to be a seminal molecular event resulting in a highly malignant neuroblastoma phenotype. PMID: 12670915 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 50: Neoplasia. 2003 Jan-Feb;5(1):53-62. Chromosomal localization of DNA amplifications in neuroblastoma tumors using cDNA microarray comparative genomic hybridization. Beheshti B, Braude I, Marrano P, Thorner P, Zielenska M, Squire JA. Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada. Conventional comparative genomic hybridization (CGH) profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available) algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb) than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1) no amplifications evident, 2) a small MYCN amplicon as the only detectable imbalance, and 3) a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma. PMID: 12659670 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 51: Cancer Genet Cytogenet. 2003 Jan 15;140(2):157-61. MYCN gain and MYCN amplification in a stage 4S neuroblastoma. Noguera R, Canete A, Pellin A, Ruiz A, Tasso M, Navarro S, Castel V, Llombart-Bosch A. Deparment of Pathology, Medical School, University of Valencia, Avda. Blasco Ibanez, 17,46010 Valencia, Spain. rosa.noguera@uv.es Stage 4S neuroblastoma is a disease associated with spontaneous regression and good survival. We present a patient whose evolution has shown the variety and complexity of this disease in infants. Biologic factors, such as ploidy, MYCN copy number, loss of 1p36, and other chromosomal gains and losses were determined. A complex pattern of genetic abnormalities, such as near-diploidy, MYCN gain (2-4 copies per haploid genome) and imbalance/deletion of 1p36 was seen in the diagnostic sample. An extensive disseminated disease after a latent period of 26 months was associated with a special genetic evolution, such a tetraploidy, MYCN amplification (2:100-500 copies), 1p36 deletion, and gain of 17q. Our results provide evidence that either the primary tumor was heterogeneous in terms of gene amplification or that amplification was acquired later on as a transition from MYCN gain. We suggest that near-di-/tetraploid 4S tumors with MYCN gain and/or deletion 1p could be progressing 4S tumors. Publication Types: Case Reports PMID: 12645655 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 52: Oncogene. 2003 Feb 20;22(7):1002-11. CHD5, a new member of the chromodomain gene family, is preferentially expressed in the nervous system. Thompson PM, Gotoh T, Kok M, White PS, Brodeur GM. Division of Oncology, the Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. Chromatin remodeling is one of the mechanisms by which gene expression is regulated developmentally. Chromatin structure is controlled at least in part by post-translational modification of histones, as well as by chromodomain proteins. We have identified a novel gene encoding a protein with chromatin remodeling, helicase and DNA-binding motifs. This gene, called CHD5, is the fifth member of the CHD gene family identified in humans. This gene is most homologous to CHD3 and CHD4, which encode proteins that are part of the nucleosome remodeling and histone deacetylation (NuRD) complex. CHD5 is preferentially expressed in total brain, fetal brain, and cerebellum. It is also moderately expressed in the adrenal gland, but expression is undetectable in almost all other tissues examined. CHD5 maps within a small region of deletion on 1p36.3 in human neuroblastomas, a common pediatric tumor. We examined a panel of neuroblastoma cell lines for CHD5 expression, which was consistently low or undetectable in all these lines. Expression was also examined in a panel of 137 primary neuroblastomas, and low expression was highly correlated with 1p deletion, MYCN amplification, advanced stage, and unfavorable histology. These findings suggest that this gene may play a role in the development of the nervous system, and it may also play a role in the pathogenesis of neural tumors. PMID: 12592387 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 53: Clin Cancer Res. 2003 Feb;9(2):812-9. Real-time analysis of tyrosine hydroxylase gene expression: a sensitive and semiquantitative marker for minimal residual disease detection of neuroblastoma. Lambooy LH, Gidding CE, van den Heuvel LP, Hulsbergen-van de Kaa CA, Ligtenberg M, Bokkerink JP, De Abreu RA. Department of Pediatrics, University Medical Center Nijmegen, 6500 HB Nijmegen, The Netherlands. PURPOSE: The purpose of this study was to establish a sensitive and semiquantitative method for the detection of minimal residual disease of neuroblastoma, the most common solid tumor in childhood. EXPERIMENTAL DESIGN: Analysis was performed on a molecular level by reverse transcription-PCR using a new, real-time detection method. We measured two genes simultaneously, tyrosine hydroxylase (TH) as the target gene and glyceraldehyde-3-phosphate dehydrogenase as a reference gene, in blood and bone marrow samples at diagnosis and after follow-up from six patients with neuroblastoma, one patient with ganglioneuroma, and one patient with ganglioneuroblastoma. RESULTS: The sensitivity of the assay was 1:10(6) peripheral WBCs. Four patients with stage IV neuroblastoma and one patient with stage III neuroblastoma were scored positive. The other stage III patient and the other two patients with ganglioneuroma and ganglioneuroblastoma followed by acute lymphoblastic leukemia, respectively, were scored negative. Control bone marrow aspirates were also negative. The TH assay is more sensitive than immunohistochemical detection, and the results of the TH assay corresponded with the results of MYCN amplification. CONCLUSIONS: The described TH assay is specific, sensitive, and semiquantitative and can be used for the detection of neuroblastoma cell involvement in bone marrow and blood at diagnosis and during therapy. Furthermore, the TH assay is a possible prognostic marker for neuroblastoma. PMID: 12576454 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 54: Oncogene. 2003 Jan 23;22(3):456-60. ID2 expression in neuroblastoma does not correlate to MYCN levels and lacks prognostic value. Vandesompele J, Edsjo A, De Preter K, Axelson H, Speleman F, Pahlman S. Center for Medical Genetics, Ghent University Hospital, Belgium. The MYCN proto-oncogene is frequently amplified in a subgroup of highly aggressive neuroblastomas. The molecular mechanism(s) by which the overexpressed MYCN contributes to an aggressive tumor cell behavior is not well understood. Recently, it was reported that the ID2 gene is a direct target for the MYCN and MYC transcription factors, and that ID2 expression and MYCN amplification correlate positively in neuroblastoma. In addition, ID2 expression was proposed as a negative prognostic parameter. As these results are of potential clinical importance, but not in agreement with our own initial observations, the putative correlation between ID2 and MYC(N) expression in neuroblastoma cell lines and tumors was reinvestigated. We found no correlation between MYCN and ID2 expression in neuroblastoma cell lines or tumor specimens. However, we did find a significant positive correlation between MYC and ID2 expressions in both MYCN-amplified and single-copy tumor specimens, and in MYCN single-copy cell lines. As previously reported, we also found an inverse correlation between MYC and MYCN expressions. Importantly, we could not confirm the reported prognostic power of ID2-expression in neuroblastoma. These data, obtained in two independent laboratories, challenge the previously proposed ID2-MYCN relation. PMID: 12545167 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 55: J Pediatr Hematol Oncol. 2003 Jan;25(1):8-13. High-risk neuroblastoma in Ontario: a report of experience from 1989 to 1995. Klaassen RJ, Trebo MM, Koplewitz BZ, Weitzman SS, Calderwood S. Department of Pediatrics, Division of Hematology/Oncology, Children's Hospital of Eastern Ontario, 401 Smyth, Ottawa, Ontario, Canada K1H 8L1. rklaassen@cheo.on.ca PURPOSE: We did a population-based study of children with high-risk neuroblastoma to determine their survival and look for factors that had an impact on survival. METHODS: We carried out a retrospective cohort study of patients diagnosed in Ontario from 1989 to 1995. 162 cases of neuroblastoma were diagnosed in the province with 70 (43%) considered high-risk: all were older than one year of age, with 15 patients classified as International Neuroblastoma Staging System (INSS) stage 3, and 55 INSS stage 4. RESULTS: Stage 3 patients did significantly better than Stage 4 patients with a 5-year overall survival of 67.7% and 22.7% respectively (P = 0.015). In stage 4 patients achieving at least a partial response to up-front therapy and surviving for at least 9.5 months after diagnosis (the median time to transplant), there was no difference in survival between the 19 transplant patients and the 17 treated with chemotherapy alone (P = 0.75). However, patients transplanted by peripheral stem cell (PSC) collection did significantly better than both the bone marrow transplantation (P = 0.002) and chemotherapy-alone group (P = 0.047). There was a significant difference in up-front chemotherapy and use of radiation in the three groups (P < 0.001 and P = 0.01 respectively), but no difference in the incidence of bone and bone marrow metastases, MYCN amplification or unfavorable histology. CONCLUSIONS: In this nonrandomized study, we found that stage 4 neuroblastoma patients alive more than 9.5 months after diagnosis, with at least a partial response to initial therapy, did significantly better with PSC transplant compared with bone marrow transplantation or chemotherapy alone. PMID: 12544767 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 56: Oncol Rep. 2003 Jan-Feb;10(1):243-7. Prognostic significance of cell proliferation in human neuroblastoma: comparison with other prognostic factors. Mejia C, Navarro S, Pellin A, Ruiz A, Castel V, Llombart-Bosch A. Department of Neurosciences, Institute of Cell Physiology, Universidad Nacional Autonoma de Mexico, Mexico City 04510, D.F. Mexico. mmejia@ifisiol.unam.mx Peripheral neuroblastic tumors (PNT), are heterogeneous neoplasms that include neuroblastoma (NB), ganglioneuroblastoma (GNB) and ganglioneuroma (GN) and present great biological heterogeneity. There are few reports analyzing PCNA and Ki-67 expression in PNT; however, controversy exists concerning the specificity of PCNA as a real proliferative marker. The objective of our study was to determine which of these cellular proliferation markers is more specific on cellular cycle and could contribute with more information on the cell cycle. We found that PCNA was expressed in NB unfavourable cases, with MYCN amplification and high mitosis-karyorrhexis-index (MKI). Whereas, Ki-67 showed statistical significance regarding cases unfavourable with intermediate and high MKI, aneuploid and stages 3 and 4. Survival showed that patients with tumor not expressing Ki-67 (MIB1) lived longer than those without PCNA (88.93 vs 74.05%). We conclude that Ki-67 expression permits reliable detection of the cellular proliferation neuroblastoma fraction and provides useful prognostic information when associated with other biological factors. PMID: 12469176 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 57: Cancer Cell. 2002 Nov;2(5):377-86. Loss of a FYN-regulated differentiation and growth arrest pathway in advanced stage neuroblastoma. Berwanger B, Hartmann O, Bergmann E, Bernard S, Nielsen D, Krause M, Kartal A, Flynn D, Wiedemeyer R, Schwab M, Schafer H, Christiansen H, Eilers M. Institute for Molecular Biology and Tumor Research, Emil-Mannkopff-Strasse 2, 35037 Marburg, Germany. Tumor stage, age of patient, and amplification of MYCN predict disease outcome in neuroblastoma. To gain insight into the underlying molecular pathways, we have obtained expression profiles from 94 primary neuroblastoma specimens. Advanced tumor stages show a characteristic expression profile that includes downregulation of multiple genes involved in signal transduction through Fyn and the actin cytoskeleton. High expression of Fyn and high Fyn kinase activity are restricted to low-stage tumors. In culture, expression of active Fyn kinase induces differentiation and growth arrest of neuroblastoma cells. Expression of Fyn predicts long-term survival independently of MYCN amplification. Amplification of MYCN correlates with deregulation of a distinct set of genes, many of which are target genes of Myc. Our data demonstrate a causal role for Fyn kinase in the genesis of neuroblastoma. PMID: 12450793 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 58: J Pediatr Hematol Oncol. 2002 Nov;24(8):613-21. Comment in: J Pediatr Hematol Oncol. 2002 Nov;24(8):608-9. Intensified chemotherapy increases the survival rates in patients with stage 4 neuroblastoma with MYCN amplification. Kaneko M, Tsuchida Y, Mugishima H, Ohnuma N, Yamamoto K, Kawa K, Iwafuchi M, Sawada T, Suita S. Study Group of Japan for Treatment of Advanced Neuroblastoma, and Department of Pediatric Surgery, University of Tsukuba, Tsukuba, Japan. PURPOSE: Patients with high-risk neuroblastoma who have multiple copies of MYCN fare much worse than do those without MYCN amplification; however, it has not been clarified whether intensified chemotherapy with or without blood stem cell transplantation can alter the extremely poor prognosis of patients with amplified MYCN. METHODS AND RESULTS: Between 1985 and 1999, 301 patients older than age 12 months with stage 4 neuroblastoma were treated. From January 1985 to February 1991, 80 patients with stage 4 neuroblastoma with and without MYCN amplification uniformly received induction chemotherapy with regimen A(1) (cyclophosphamide 1,200 mg/m(2) and vincristine 1.5 mg/m(2) on day 1, tetra-hydropyranyl [THP]-Adriamycin 40 mg/m(2) on day 3, and cisplatin 90 mg/m(2) on day 5). Among 22 patients with MYCN amplification, nine (40.9%) achieved a complete remission and seven (31.8%) underwent stem cell transplantation. Of 58 patients without MYCN amplification, 43 (74.1%) achieved a complete remission and 14 (24.1%) underwent stem cell transplantation. The 5-year relapse-free survival rates were 23.2% for stage 4 patients with MYCN amplification and 33.3% for those without MYCN amplification (P = 0.029); the 5-year overall survival rates were 32.8% for stage 4 patients with MYCN amplification and 42.8% for those without MYCN amplification (P > 0.05). From March 1991 to June 1998, patients with stage 4 neuroblastoma who had 10 or more copies of MYCN were treated with regimen A(3) (cyclophosphamide 1,200 mg/m(2) per day on days 1 and 2, THP-Adriamycin 40 mg/m(2) on day 3, etoposide 100 mg/m(2) per day on days 1 to 5, and cisplatin 25 mg/m(2) per day on days 1 to 5); those with fewer than 10 copies of MYCN received regimen new A (cyclophosphamide 1,200 mg/m on day 1, THP-Adriamycin 40 mg/m on day 3, etoposide 100 mg/m per day on days 1 to 5, and cisplatin 90 mg/m on day 5), which is similar in intensity to regimen A. Among 88 patients with MYCN amplification, 63 (71.6%) achieved a complete remission and 63 (71.68%) underwent stem cell transplantation. Of 133 patients without MYCN amplification, 93 (69.9%) achieved a complete remission and 71 (53.4%) underwent stem cell transplantation. The 5-year relapse-free survival rates were 36.0% for stage 4 patients with MYCN amplification and 32.2% for those without MYCN amplification (P > 0.05), the 5-year overall survival rates were 34.0% for stage 4 patients with MYCN amplification and 38.9% for those without MYCN amplification (P > 0.05). The difference in relapse-free survival rates was significantly different (P = 0.003) between patients with MYCN-amplified tumor treated before (regimen A(1)) versus after 1991 (regimen A(3)). CONCLUSIONS: With the use of the more intensive induction regimen A plus blood stem cell transplantation for MYCN-amplified patients, survival curves for those with or without MYCN amplification now appear similar. Higher doses of chemotherapy may ameliorate the effect of MYCN amplification in patients with high-risk neuroblastoma. Publication Types: Multicenter Study PMID: 12439032 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 59: J Pediatr Hematol Oncol. 2002 Nov;24(8):608-9. Comment on: J Pediatr Hematol Oncol. 2002 Nov;24(8):613-21. Commentary on Kaneko et al.: Intensified chemotherapy increases the survival rates in patients with stage 4 neuroblastoma with MYCN amplification. Brodeur GM. Publication Types: Comment Editorial PMID: 12439030 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 60: Surgery. 2002 Aug;132(2):232-8. Importance of Sp1 consensus motifs in the MYCN promoter. Inge TH, Casson LK, Priebe W, Trent JO, Georgeson KE, Miller DM, Bates PJ. Children's Hospital Research Foundation and Department of Pediatric Surgery, Cincinnati Children's Hospital Medical Center, Ohio 45229,USA. BACKGROUND: MYCN (N-myc) amplification in neuroblastoma is associated with poor clinical outcome. Factors that regulate MYCN expression have not been elucidated. MYCN is considered a TATA-less promoter, whereas significant promoter activity resides within 160 bp 5' of the major transcription start site. This region contains two GC-rich motifs and a CT box, regions for potential transcription factor interaction. METHODS: To characterize DNA-protein interactions in this region of the MYCN promoter, electrophoretic mobility shift assays, and promoter-reporter were used. RESULTS: A MYCN promoter fragment was incubated with HeLa nuclear extract, with or without competitors. Three major protein/DNA complexes were formed. Formation of 2 complexes could be inhibited by unlabeled Sp1 consensus duplex and by the Sp1 site-specific drug WP631. Purified Sp1 protein produced a complex similar to that formed with HeLa extract. To determine whether these DNA/protein interactions could be blocked in a sequence-specific fashion, a triplex forming oligonucleotide (TFO) was used. This TFO was designed to bind in the major groove of the promoter, covering the CT-box (putative Sp1 binding) motif. When triplex formation was followed by addition of nuclear extract, protein binding was indeed inhibited. Functional significance of this inhibition was tested with pE/Bnmyc-luc, a promoter-reporter plasmid containing the human MYCN promoter driving luciferase expression. Incubation with TFO, but not control oligodeoxynucleotides, completely inhibited luciferase activity. CONCLUSIONS: These data suggest that protein binding does occur in regions of the MYCN promoter containing GC and CT box elements and that this interaction is important for MYCN promoter activity. By inference, these data also suggest that the proteins that bind in this region are Sp1 family members. PMID: 12219017 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 61: Biochem Biophys Res Commun. 2002 Aug 9;296(1):152-5. Gene amplification profiling of esophageal squamous cell carcinomas by DNA array CGH. Ishizuka T, Tanabe C, Sakamoto H, Aoyagi K, Maekawa M, Matsukura N, Tokunaga A, Tajiri T, Yoshida T, Terada M, Sasaki H. Genetics Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, 104-0045, Tokyo, Japan. Gene amplification is one of the basic mechanisms that lead to overexpression of oncogenes. DNA array comparative genomic hybridization (CGH) has great potential for comprehensive analysis of both a relative gene-copy number and altered chromosomal regions in cancers, which enables us to identify new amplified genes and unstable chromosomal loci. We examined the amplification status in 32 esophageal squamous cell carcinomas (ESCCs) and 13 ESCC cell lines on 51 frequently amplified loci in a variety of cancers by both DNA array CGH and Southern blot analyses. The 1p34 locus containing MYCL1, 2p24 (MYCN), 7p12 (EGFR), and 12q14 (MDM2) were amplified in one of the 32 cases (3%), and the 17q12 locus (ERBB2) and 8p11 (FGFR1) in two of the 32 cases (6%), while only the 11q13 locus (Cyclin D1, FGF4, and EMS1) was frequently amplified (28%, 9/32), demonstrating this locus to be a major target in ESCCs. One locus, 8q24 (c-MYC) was found to be amplified only in the cell lines. Eight out of 51 loci (15.7%) were found to be amplified in at least one of the 32 primary ESCCs or the 13 ESCC cell lines, suggesting that chromosomal loci frequently amplified in a type of human cancer may also be amplified in other types of cancers. This paper is the first report of an application of DNA array CGH to ESCCs. PMID: 12147242 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 62: Cancer Lett. 2002 Oct 28;184(2):127-47. Genetic parameters of neuroblastomas. Westermann F, Schwab M. Department of Cytogenetics (H0400), German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. f.westermann@dkfz.de Neuroblastoma is a malignant childhood tumor of migrating neuroectodermal cells derived from the neural crest and destined for the adrenal medulla and the sympathetic nervous system. The biological behavior of neuroblastomas is extremely variable and in some respects unique. Neuroblastomas tend to regress spontaneously in a portion of infants or to differentiate into a benign ganglioneuroma in some older patients. Unfortunately, in the majority of patients neuroblastoma is metastatic at the time of diagnosis, and it usually undergoes rapid progression with a fatal outcome. The mechanisms leading to this diverse clinical behavior of neuroblastomas are largely unclear. From the analysis of tumors at the cytogenetic and molecular level non-random genetic changes have been identified, including ploidy changes, amplification of the oncogene MYCN, deletions of chromosome 1p, gains of chromosome arm 17q, and deletions of 11q as well as of other genomic regions that allow tumors to be classified into subsets with distinct biological features and clinical behavior. MYCN status is widely accepted for therapy stratification. Additional genetic parameters are currently under investigation to refine risk assessment, but so far the molecular monitoring tools for prediction of therapy response and disease outcome are still incomplete. This should lead to more risk-adapted therapies according to the clinical-genetic parameters by which individual tumors are characterized. This review aims at discussing the role of genomic changes in neuroblastomas of diverse biological and clinical types. Publication Types: Review PMID: 12127685 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 63: Ann N Y Acad Sci. 2002 Jun;963:63-73. Amplified MYCN in human neuroblastoma: paradigm for the translation of molecular genetics to clinical oncology. Schwab M. Division of Cytogenetics, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. m.schwab@dkfz.de Increase in the dosage of cellular oncogenes by DNA amplification is a frequent genetic alteration of cancer cells. In neuroblastoma, amplification of the gene MYCN has been associated with aggressively growing cancers and is an indicator for poor prognosis. MYCN amplification is of predictive value in identifying patients with neuroblastoma who require specific therapeutic regimens and those who do not benefit from chemotherapy. PMID: 12095930 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 64: Genes Chromosomes Cancer. 2002 Jul;34(3):299-305. Fluorescence in situ hybridization analyses of chromosome band 1p36 in neuroblastoma detect two classes of alterations. Spitz R, Hero B, Westermann F, Ernestus K, Schwab M, Berthold F. University Children's Hospital, Department of Pediatric Oncology, Cologne, Germany. Ruediger.Spitz@medizin.uni-koeln.de Chromosomal alterations in 1p36 were investigated in 196 neuroblastoma tumors using fluorescence in situ hybridization. Additionally, by using the same technique, it was determined whether MYCN was amplified in 149 of these. The most frequent finding was a deletion in 1p36, leading to monosomy of this region (29 cases, 15%). Furthermore, we found tumors with at least two intact copies of chromosome 1 and additional 1p36-deleted copies. Altogether, 21 tumors (11%) displayed this imbalance of 1p36. Similar to the cases with deletion, imbalances were predominantly found in stage 4 tumors (81%), and they were significantly associated with an increased patient age (P = 0.01). Nearly all 1p-deleted tumors showed amplification of MYCN (24/27 analyzed samples, 89%), whereas only 8 of 21 (38%) with imbalance did. Eight cases with imbalance were investigated for loss of heterozygosity (LOH) using microsatellite markers in 1p35-36. Only 4 displayed 1p36 LOH, whereas the remaining 4 were heterozygous. Both patients with deletion of 1p and with imbalance had a poor outcome [3-year rate of event-free-survival (EFS): 33 +/- 15% and 41 +/- 15%], which was significantly worse compared to the outcome of patients without 1p alterations (3-year EFS: 70 +/- 5%; P = 0.01 and P = 0.0059). We conclude that besides monosomic short arm deletions, imbalance of 1p36 is a strong marker of a poor prognosis in neuroblastoma and not necessarily associated with MYCN amplification and LOH. Copyright 2002 Wiley-Liss, Inc. PMID: 12007190 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 65: Arch Pathol Lab Med. 2002 May;126(5):540-4. MYCC and MYCN oncogene amplification in medulloblastoma. A fluorescence in situ hybridization study on paraffin sections from the Children's Oncology Group. Aldosari N, Bigner SH, Burger PC, Becker L, Kepner JL, Friedman HS, McLendon RE. Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA. 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. PMID: 11958658 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 66: Br J Cancer. 2002 Apr 8;86(7):1110-6. Experience with International Neuroblastoma Staging System and Pathology Classification. Ikeda H, Iehara T, Tsuchida Y, Kaneko M, Hata J, Naito H, Iwafuchi M, Ohnuma N, Mugishima H, Toyoda Y, Hamazaki M, Mimaya J, Kondo S, Kawa K, Okada A, Hiyama E, Suita S, Takamatsu H. Department of Pediatric Surgery, Dokkyo University School of Medicine, Koshigaya Hospital, 2-1-50 Minami-Koshigaya, Koshigaya, Saitama 343-8555, Japan. The International Neuroblastoma Staging System and Pathology Classification were proposed in 1988 and in 1999, respectively, but their clinical value has not yet been fully studied in new patients. Six hundred and forty-four patients with neuroblastoma treated between January 1995 and December 1999 were analysed by these classifications. The 4-year overall survival rate of patients <12 months of age with INSS stages 1, 2A, 2B, 3 and 4S disease was 98.5%, which was significantly higher than the 73.1% rate in stage 4 patients <12 months (P<0.0001). When patients were > or = 12 months, the 4-year overall survival rate of patients with neuroblastoma at 1, 2A, 2B and 3 stages was 100% and that of patients at stage 4 was 48.5% (P<0.0001). As to the International Neuroblastoma Pathology Classification histology, the 4-year overall survival rate was 98.8% in patients with favourable histology and 60.7% in those with unfavourable histology in the <12 months group (P<0.0001). In the > or = 12 months group, the 4-year oral survival of patients with favourable histology was 95.3% and that of patients with unfavourable histology was 50.6% (P<0.0001). Among biological factors, MYCN amplification, DNA diploidy and 1p deletions were significantly associated with poor prognosis in patients <12 months, as were MYCN amplification and DNA diploidy in patients > or = 12 months of age. Multivariate analysis showed that the INSS stage (stage 4 vs other stages) and International Neuroblastoma Pathology Classification histology (unfavourable vs favourable) were significantly and independently associated with the survival of patients undergoing treatment, stratified by age, stage and MYCN amplification (P=0.0002 and P=0.0051, respectively). PMID: 11953858 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 67: J Pathol. 2002 Apr;196(4):450-8. MYCN expression in human rhabdomyosarcoma cell lines and tumour samples. Toffolatti L, Frascella E, Ninfo V, Gambini C, Forni M, Carli M, Rosolen A. Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Universita di Padova, Padova, Italy. The MYCN oncogene encodes a phosphoprotein that acts as a transcription factor and is involved in the regulation of cell proliferation and differentiation in normal as well as in cancer cells.MYCN amplification and expression have been reported in various tumours, including neuroblastoma and lung cancer, but little is known about its expression in human rhabdomyosarcoma. MYCN expression and amplification were studied in five alveolar and five embryonal rhabdomyosarcoma cell lines and in 19 tumour biopsies. All the cell lines studied expressed MYCN RNA, as demonstrated by northern blot analysis and RT-PCR, but the oncogene was amplified in only one. Similarly, MYCN protein was detected in all cell lines by western blot analysis, with higher levels of expression in alveolar than in embryonal rhabdomyosarcoma cells. RT-PCR analysis of tumour samples demonstrated 18/19 cases positive for MYCN RNA. Although MYCN expression was higher in alveolar than in embryonal rhabdomyosarcoma cell lines, no clear relationship between histology and level of MYCN expression could be established in this tumour series. These data suggest that MYCN expression is a common feature of rhabdomyosarcoma, independent of gene amplification and without a clear relationship with specific histological and clinical features. Copyright 2002 John Wiley & Sons, Ltd. PMID: 11920742 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 68: Pediatr Dev Pathol. 2002 Mar-Apr;5(2):190-9. Hormonal activity may predict aggressive behavior in neuroblastoma. Zambrano E, Reyes-Mugica M. Department of Pathology, Yale University School of Medicine, 310 Cedar Street, Lauder Hall, CB20, P.O. Box 208023, New Haven, CT 06520-8023, USA. Overproduction of catecholamines (dopamine [DA], norepinephrine [NE]) and their metabolites (homovanillic [HVA] and vanillylmandelic [VMA] acids) characterizes neuroblastoma (NB). In previous studies, increased urinary DA/NE, and DA/VMA ratios have been associated with poor prognosis, whereas low DA/NE ratios have been associated with longer disease-free survival. Higher urinary VMA, HVA, and NE levels have been found in association with low MYCN amplification, in contrast to cases with high MYCN amplification in which normal levels have been found. It is then believed that an "immature" catecholamine pattern indicates poor prognosis. We correlated urinary DA, NE, VMA, and HVA levels with age, clinical tumor stage, histological features (favorable [FH]/unfavorable [UH]) and MYCN status of 33 patients with NB. DA/VMA and DA/HVA ratios were also calculated. Wilcoxon rank sum and chi-squared tests were performed to determine statistical significance. Eighty-eight percent (15/17) of stage 3-4 cases had DA levels >2 times the upper limit of normal, but only 8% (1/12) of stage 1-2 cases had DA levels twice the upper limit of normal. In 61% (11/18) of stage 3-4 cases, the VMA level was >10 times the upper limit of normal, in contrast to stage 1-2 cases, in which only one patient (1/15) had a VMA level >10 times the upper limit of normal. Similar findings were obtained with urinary HVA and NE. Patients older than 12 months of age at diagnosis also had higher urinary levels of DA, VMA, HVA, and NE than those of patients younger than 12 months of age at diagnosis. Eighty-two percent (14/17) of stage 3-4 cases had DA/VMA ratios <0.78, with the other 18% (3/17) showing ratios between 1.4 and 8.82 (all stage 4 and >12 months of age). In contrast, all stage 1-2 cases ((12)) had ratios <1.4. All (12/12) non- MYCN-amplified cases had DA/VMA ratios <1.4 (0.06-0.84), while one MYCN-amplified case (1/3) had a ratio of 8.82; the other two MYCN-amplified cases had DA/VMA ratios of 0.09-0.11. Twenty-nine percent (2/7) of cases with UH had a DA/VMA ratio >1.4, but in all FH cases (14/14) the DA/VMA ratio was <1.4 (0.08-0.084). Similar to previous studies, we found that aggressive NB is associated with higher urinary levels of DA, VMA, HVA, and NE. We also confirmed the previous observation that there appears to be a subset of NB in which a possible blockade in DA metabolism is associated with poor prognostic features (>12 months, stage 4, UH, and MYCN amplification). A seemingly novel observation in our study is that all high DA/HVA and DA/VMA ratios were obtained in stage 4 tumors, suggesting an association between the inability to metabolize DA and the acquisition of metastatic potential. On the basis of our results, we would like to emphasize the importance of determining not only DA, HVA, and VMA urinary levels, to support the diagnosis of NB, but also DA/HVA and DA/VMA ratios as a rapid initial assessment of prognosis in these patients. PMID: 11910515 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 69: Oncogene. 2002 Mar 14;21(12):1848-58. Caspase-9 and Apaf-1 are expressed and functionally active in human neuroblastoma tumor cell lines with 1p36 LOH and amplified MYCN. Teitz T, Wei T, Liu D, Valentine V, Valentine M, Grenet J, Lahti JM, Kidd VJ. Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, TN 38105, USA. Important roles have been suggested for caspase-8, caspase-9 and Apaf-1 in controlling tumor development and their sensitivity to chemotherapeutic agents. Methylation and deletion of Apaf-1 and CASP8 results in the loss of their expression in melanoma and neuroblastoma, respectively, while CASP9 localization to 1p36.1 suggests it is a good candidate tumor suppressor. The status of CASP9 and Apaf-1 expression in numerous neuroblastoma cell lines with/without amplified MYCN and chromosome 1p36 loss-of-heterozygosity (LOH) was therefore examined to test the hypothesis that one or both of these genes are tumor suppressors in neuroblastoma. Although CASP9 is included in the region encompassing 1p36 LOH in all neuroblastoma cell lines examined, the remaining CASP9 allele(s) express a functional caspase-9 enzyme. Apaf-1 is also expressed in all neuroblastoma tumor cell lines examined. Thus, the CASP9 or Apaf-1 genes do not appear to function as tumor suppressors in MYCN amplified neuroblastomas. However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria. This suggests that a second, smaller sub-group of MYCN amplified neuroblastoma tumors exists with defect(s) in apoptotic signaling components upstream of caspase-9 and Apaf-1. Since no consistent differences in Bcl-2, Bcl-x(L) or Bax expression were seen in the STS- and radiation-resistant neuroblastomas, it suggests that a unique mitochondrial signaling factor(s) is responsible for the defect in cytochrome c release in this sub-group of tumors. PMID: 11896617 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 70: Cancer Res. 2002 Feb 15;62(4):1123-8. Minichromosome maintenance protein MCM7 is a direct target of the MYCN transcription factor in neuroblastoma. Shohet JM, Hicks MJ, Plon SE, Burlingame SM, Stuart S, Chen SY, Brenner MK, Nuchtern JG. Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA. The MYCN oncogene is amplified in approximately 25% of neuroblastoma tumors and is the most significant negative prognostic factor. The direct transcriptional targets of MYCN in MYCN-amplified tumors have not been defined. Microarray analysis of RNA from neuroblastoma primary cell cultures revealed 10-fold higher MCM7 expression in MYCN-amplified versus nonamplified tumors. MCM7 is an essential component of DNA replication licensing factor, a hexameric protein complex that regulates DNA synthesis during the cell cycle, preventing rereplication and ensuring maintenance of DNA euploidy. Additional experiments demonstrated markedly increased expression of MCM7 RNA and protein in MYCN-amplified neuroblastoma tumors and cell lines. Induction of MYCN in conditional cell lines results in increased expression of endogenous MCM7 mRNA and a 3-fold increase in protein levels. In addition, luciferase activity from MCM7 promoter/luciferase gene reporter constructs was significantly increased under MYCN-induced conditions. Specific electrophoretic mobility shifts of MCM7 promoter sequences are detected in extracts of MYCN-amplified cells. These findings demonstrate that in neuroblastoma, the MYCN oncogene directly activates genes required for DNA replication, and this may contribute to neoplastic transformation of these MYCN-amplified tumors. PMID: 11861392 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 71: Cancer. 2002 Feb 1;94(3):854-61. Proliferation marker KI-S5 discriminates between favorable and adverse prognosis in advanced stages of neuroblastoma with and without MYCN amplification. Krams M, Hero B, Berthold F, Parwaresch R, Harms D, Rudolph P. Department of Pathology, University of Kiel, Kiel, Germany. mkrams@path.uni-kiel.de BACKGROUND: The biologic behavior of neuroblastoma is notoriously variable, and even carefully elaborated prognostic models fail to predict the clinical course in a portion of cases. Because the proliferative activity is determined by the sum of all molecular imbalances that influence cell cycling, the authors investigated the potential prognostic relevance of the tumor growth fraction in neuroblastoma. METHODS: A retrospective analysis was conducted on a cohort of 161 neuroblastoma patients with a median follow-up period of 72.8 months. Tumors were classified according to Hughes typing and grading criteria. The proliferative index (PI) was assessed immunohistochemically on archival biopsy specimens using monoclonal antibody Ki-S5 (Ki-67), and the MYCN status was determined by means of Southern blot analysis. RESULTS: The PI, MYCN status, International Neuroblastoma Staging System (INSS) stage, International Neuroblastoma Pathology Classification grade, Hughes grade, and the patients' age at diagnosis were all found to be significant predictors of event free survival by univariate Kaplan-Meier analysis. However, the PI identified prognostically distinct subsets in higher tumor stages and Grade 2 and 3 neuroblastomas as well as tumors with unfavorable histology, and enabled risk stratification in tumors with and without MYCN amplification (P = 0.034 and 0.002, respectively). Multivariate Cox regression analysis selected INSS stage (relative risk [RR], 4.05; P < 0.0001) and the PI (RR, 2.49; P = 0.007) as the sole independent prognostic indicators, whereas MYCN entered the selection only after exclusion of the PI. CONCLUSIONS: It emerges that the PI as a single factor has greater predictive power than the MYCN status. Proliferation measurements therefore might significantly improve the accuracy of current prognostic models for neuroblastoma. Copyright 2002 American Cancer Society. DOI 10.1002/cncr.10256 PMID: 11857322 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 72: Mod Pathol. 2002 Feb;15(2):159-66. Quantification of MYCN, DDX1, and NAG gene copy number in neuroblastoma using a real-time quantitative PCR assay. De Preter K, Speleman F, Combaret V, Lunec J, Laureys G, Eussen BH, Francotte N, Board J, Pearson AD, De Paepe A, Van Roy N, Vandesompele J. Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium. Amplification of the proto-oncogene MYCN is a strong adverse prognostic factor in neuroblastoma patients in all tumor stages. The status of the MYCN gene has become an important factor in clinical decision making and therapy stratification. Consequently, fast and accurate assessment of MYCN gene copy number is of the utmost importance and the use of two independent methods to determine MYCN status is recommended. For these reasons we have developed and evaluated a real-time quantitative PCR (Q-PCR) assay as an alternative for time-consuming Southern blot analysis (SB), and as a second independent technique in parallel with fluorescence in situ hybridization (FISH) analysis. Advantages of Q-PCR are a large dynamic range of quantification, no requirement for post-PCR sample handling and the need for very small amounts of starting material. The accuracy of the assay was illustrated by measurement of MYCN single gene copy changes in DNA samples of two patients with 2p deletion and duplication, respectively. Two different detection chemistries i.e., a sequence specific TaqMan probe and a generic DNA binding dye SYBR Green I were evaluated and shown to yield similar results. Also, two different calculation methods for copy number determination were used i.e., the kinetic method and the comparative C(T) method, and shown to be equivalent. In total, 175 neuroblastoma samples with known MYCN status, as determined by FISH and/or SB, were examined. Q-PCR data were highly concordant with FISH and SB data. In addition to MYCN copy number evaluation, DDX1 and NAG gene copy numbers were determined using a similar Q-PCR strategy. Survival analysis pointed out that DDX1 and/or NAG amplification has no additional adverse effect on prognosis. PMID: 11850545 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 73: Med Pediatr Oncol. 2002 Feb;38(2):109-11. Comment in: Med Pediatr Oncol. 2002 Feb;38(2):112-3. Neuroblastoma with focal MYCN amplification and bone marrow infiltration: a staging and treatment dilemma. Kerbl R, Ambros IM, Ambros PF, Lackner H, Dornbusch HJ, Urban CE. Department of Pediatrics, Division of Hematology and Oncology, University of Graz, Graz, Austria. reinhold.kerbl@kfunigraz.ac.at Publication Types: Case Reports PMID: 11813175 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 74: Cancer. 2001 Nov 15;92(10):2699-708. Histopathology (International Neuroblastoma Pathology Classification) and MYCN status in patients with peripheral neuroblastic tumors: a report from the Children's Cancer Group. Goto S, Umehara S, Gerbing RB, Stram DO, Brodeur GM, Seeger RC, Lukens JN, Matthay KK, Shimada H. Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles, and Keck School of Medicine, University of Southern California, Los Angeles, California. BACKGROUND: The International Neuroblastoma Pathology Classification (International Classification), which was established in 1999, is significant prognostically and is relevant biologically for the evaluation and analysis of patients with neuroblastic tumors (NTs). MYCN amplification is a known molecular marker for aggressive progression of NTs. These have been used together as important prognostic factors to define risk groups for patient stratification and protocol assignment. METHODS: A total of 628 NTs (535 neuroblastomas [NBs]); 21 ganglioneuroblastoma, intermixed [GNBi]; 9 ganglioneuromas [GN]; and 63 ganglioneuroblastoma, nodular [GNBn]) from the Children's Cancer Group studies were evaluated histologically (favorable histology [FH] tumors vs. unfavorable histology [UH] tumors) according to the International Classification and were tested molecularly for MYCN status (amplified vs. nonamplified). Four tumor subsets (FH-nonamplified, FH-amplified, UH-nonamplified, and UH-amplified) were defined by histopathology and MYCN status, and their prognostic effects were analyzed. Detailed analysis between morphologic indicators (grade of neuroblastic differentiation and mitosis-karyorrhexis index [MKI]) and MYCN status was done by using tumors in the NB category. RESULTS: There were 339 FH-nonamplified tumors (5-year event free survival [EFS], 92.1%); 8 FH-amplified tumors (EFS, 37.5%); 172 UH-nonamplified tumors (EFS, 40.9%); and 109 UH-amplified tumors (EFS, 15.0%). The prognostic effects on patients with tumors in the four subsets were independent from the factors of patient age and disease stage (P < 0.0001). MYCN amplification was seen almost exclusively in tumors of the NB category, and no patients with tumors in either the GNBi category or in the GN category and only two patients with tumors in the GNBn category had amplified MYCN. Among the patients with tumors in the NB category, patients with FH-nonamplified tumors (309 patients) had an excellent prognosis, and patients with UH-amplified tumors (107 patients) had the poorest clinical outcome in any age group. The prognosis for children with UH-nonamplified tumors (111 patients) was poor when they were diagnosed at age > 1.5 years. It was also noted that patients with UH-amplified tumors (median age, 2.14 years) were diagnosed at a significantly younger age compared with the patients with UH-nonamplified tumors (median age, 3.55 years). Histologically, MYCN-amplified tumors lacked neuroblastic differentiation regardless of the age of patients. MYCN amplification also was linked generally to increased mitotic and karyorrhectic activities. However, MKI classes in patients with MYCN-amplified tumors varied significantly, depending on the age at diagnosis, and younger patients had higher MKI classes. CONCLUSIONS: The combination of histopathologic evaluation and MYCN status distinguishes four clinical and biologic tumor subsets in patients with NTs. MYCN amplification seems to be the powerful driving force for preventing cellular differentiation regardless of patient age and for increasing mitotic and karyorrhectic activities in an age dependent manner. Copyright 2001 American Cancer Society. PMID: 11745206 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 75: J Clin Pathol. 2001 Dec;54(12):897-910. Neuroblastoma tumour genetics: clinical and biological aspects. Bown N. School of Biochemistry and Genetics, University of Newcastle upon Tyne/Northern Genetics Service, Royal Victoria Infirmary, 19/20 Claremont Place, Newcastle upon Tyne NE2 4AA, UK. Nick.Brown@ncl.ac.uk Neuroblastoma tumour cells show complex combinations of acquired genetic aberrations, including ploidy changes, deletions of chromosome arms 1p and 11q, amplification of the MYCN oncogene, and-most frequently-gains of chromosome arm 17q. Despite intensive investigation, the fundamental role of these features in neuroblastoma initiation and progression remains to be understood. Nonetheless, great progress has been made in relating tumour genetic abnormalities to tumour behaviour and to clinical outcome; indeed, neuroblastoma provides a paradigm for the clinical importance of tumour genetic abnormalities. Knowledge of MYCN status is increasingly being used in treatment decisions for individual children, and the clinical value of 1p and 17q data as adjuncts or refinements in risk stratification is under active investigation. Reliable detection of these molecular cytogenetic features should be regarded as mandatory for all new cases at presentation. Publication Types: Review PMID: 11729208 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 76: J Biol Chem. 2002 Jan 18;277(3):1967-73. Epub 2001 Nov 15. HuD, a neuronal-specific RNA-binding protein, increases the in vivo stability of MYCN RNA. Manohar CF, Short ML, Nguyen A, Nguyen NN, Chagnovich D, Yang Q, Cohn SL. Department of Pediatrics and The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA. MYCN amplification and consequent deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma. Enhanced expression of MYCN confers growth potential to neuroblastoma cells, and a direct link between MYCN expression and the development of neuroblastoma has been demonstrated in transgenic mice studies. Although the molecular pathways underlying the regulation of MYCN have not been fully elucidated, post-transcriptional mechanisms appear to be important. Previously, we reported that an embryonic lethal abnormal vision-like (ELAV) protein binds with high specificity to at least two AU-rich elements within the MYCN 3'-untranslated region. In this study, we characterized the ability of cis-acting elements within the MYCN 3'-untranslated region to destabilize mRNA in cells and examined the functional consequences of its interactions with the ELAV protein HuD. We show that at least 4 cis-acting elements within the MYCN 3'-untranslated region are able to signal the degradation of stable heterologous mRNA. Ectopic overexpression of HuD dramatically inhibits RNA decay mediated by the full-length MYCN 3'-untranslated region and cis-acting destabilizing elements that harbor HuD binding sites in vivo. HuD may contribute to the malignant phenotype of neuroblastoma cells by stabilizing MYCN mRNA, thereby enhancing steady-state levels of expression of this oncogene. PMID: 11711535 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 77: Am J Pathol. 2001 Nov;159(5):1925-32. Regulation of telomerase activity by alternate splicing of human telomerase reverse transcriptase mRNA in a subset of neuroblastomas. Krams M, Claviez A, Heidorn K, Krupp G, Parwaresch R, Harms D, Rudolph P. Department of Pathology, University of Kiel, Kiel, Germany. mkrams@path.uni-kiel.de It has been proposed that the regulation of telomerase takes place at the transcriptional level, the expression of the catalytic subunit human telomerase reverse transcriptase (hTERT) being crucial for telomerase activity (TA). Recently, differential splicing of hTERT mRNA has been demonstrated in various tissues during embryonal development, and it has been suggested that only full-length transcripts translate into functionally active telomerase. With this in view, we analyzed the different hTERT transcripts by reverse transcriptase-polymerase chain reaction in neuroblastic tumors and compared the results with the TA, the tumor growth fraction, and the MYCN status. In a series of 38 neuroblastic tumors, high TA and full-length hTERT transcripts were found in nine samples, whereas nine samples showed absence of both enzymatic activity and hTERT transcripts. Interestingly, in another eight samples, low or absent TA coincided with a lack of full-length hTERT transcripts. Eleven samples contained hTERT transcripts with low or undetectable TA and one sample had low TA but no hTERT transcripts. TA correlated with MYCN amplification and was weakly associated with the proliferative activity. Moreover, a significant correlation with tumor progression was observed. Our findings point at a posttranscriptional regulation of TA in a subset of neuroblastic tumors. Because high TA was detected only in tumors with full-length hTERT transcripts, reverse transcriptase-polymerase chain reaction analysis of archival neuroblastic tumor samples might help to appraise the malignant potential in individual cases. PMID: 11696453 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 78: Cancer Genet Cytogenet. 2001 Oct 15;130(2):133-40. Molecular cytogenetic characterization of two non-MYCN amplified neuroblastoma cell lines with complex t(11;17). McConville CM, Dyer S, Rees SA, Luttikhuis ME, McMullan DJ, Vickers SJ, Ramani P, Redfern D, Morland BJ. Division of Medical and Molecular Genetics, University of Birmingham, Birmingham B15 2TT, UK. c.mcconville@bham.ac.uk The pediatric tumor neuroblastoma is characterized by a very variable, and at times unpredictable, pattern of clinical behavior, ranging from a benign localized tumor to an aggressive malignancy with poor prognosis. Standard clinical and pathological assessments do not always differentiate reliably between tumor subtypes and, therefore, genetic markers are now playing an increasingly important role in treatment decisions. MYCN oncogene amplification, for example, provides a useful marker of poor prognosis. However, less than one-half of all patients who present with, or who later develop, metastatic disease show MYCN amplification. Consequently, the identification of characteristic patterns of genetic alteration in the remaining tumors is of importance. In this report, we describe two new cell lines that we have established from metastatic, non-MYCN amplified, advanced stage neuroblastomas. These cell lines show a number of features in common, including unbalanced translocation between 11q and 17q, loss of 3p, 4p and 11q and gain of 17q. Therefore, they provide a valuable resource for the characterization of genetic pathways leading to aggressive tumor growth in non-MYCN amplified neuroblastomas. Publication Types: Case Reports PMID: 11675134 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 79: Oncogene. 2001 Sep 13;20(41):5913-9. Functional interaction of Yaf2 with the central region of MycN. Bannasch D, Madge B, Schwab M. Division of Cytogenetics-H0400, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. MYCN is often amplified in advanced-stage neuroblastomas with the consequence of enhanced MycN protein expression. By employing the yeast two-hybrid system we found that Yaf2 binds to the central region of MycN. Binding was also seen in vitro and in vivo. Ectopically expressed Yaf2, like MycN, is localized in the nuclei of neuroblastoma cells. Endogenous Yaf2 is expressed in all three tested neuroblastoma cell lines, all of which also express MycN. Yaf2 was able to enhance MycN-mediated transactivation from an E-box promoter, deletion of the Yaf2 binding region in MycN abrogates this effect. Thus, the binding of Yaf2 to the central region of MycN is functional in mammalian cells. PMID: 11593398 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 80: Cancer Res. 2001 Sep 15;61(18):6892-8. Comment in: Cancer Res. 2002 May 15;62(10):2986-7; author reply 2988-9. Cancer Res. 2002 May 15;62(10):2987-8; author reply 2988-9. Neuroblastic and Schwannian stromal cells of neuroblastoma are derived from a tumoral progenitor cell. Mora J, Cheung NK, Juan G, Illei P, Cheung I, Akram M, Chi S, Ladanyi M, Cordon-Cardo C, Gerald WL. Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. The coexistence of neuroblastic and Schwannian stromal (SS) cells in differentiating neuroblastoma (NB), and derivation of Schwannian-like cells from neuroblastic clones in vitro, were accepted previously as evidence of a common pluripotent tumor stem line. This paradigm was challenged when SS cells were suggested to be reactive in nature. The advent of microdissection techniques, PCR-based allelic analysis, and in situ fluorescent cytometry made possible the analysis of pure cell populations in fresh surgical specimens, allowing unequivocal determination of clonal origins of various cell subtypes. To overcome the complexity and heterogeneity of three-dimensional tissue structure, we used: (a) Laser-Capture Microdissection to obtain histologically homogeneous cell subtype populations for allelotype analysis at chromosomes 1p36, 11q23, 14q32, and 17q and study of MYCN copy number; (b) multiparametric analysis by Laser-Scanning Cytometry of morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor fluorescence in situ hybridization on touch imprints from manually microdissected neuroblast and stroma-rich areas. Histologically distinct SS and neuroblastic cells isolated by Laser-Capture Microdissection had the same genetic composition in 27 of 28 NB analyzed by allelic imbalance and gene copy number. In all 20 cases studied by Laser-Scanning Cytometry, SS cells identified by morphology and S-100 immunostaining had identical DNA content and GD2-staining pattern as their neuroblastic counterparts. In 7 cases, fluorescence in situ hybridization demonstrated the same chromosomal makeup for SS and neuroblastic cells. These results provide unequivocal evidence that neuroblastic and SS cells in NB are derived from genetically identical neoplastic cells and support the classical paradigm that NB arises from tumoral cells capable of development along multiple lineages. PMID: 11559566 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 81: Klin Padiatr. 2001 Jul-Aug;213(4):186-90. Traditional and emerging molecular markers in neuroblastoma prognosis: the good, the bad and the ugly. Poremba C, Hero B, Goertz HG, Scheel C, Wai D, Schaefer KL, Christiansen H, Berthold F, Juergens H, Boecker W, Dockhorn-Dworniczak B. Gerhard-Domagk-Institute of Pathology, Westfalische Wilhelms-University, Domagkstrasse 17, 48149 Munster, Germany. poremba@uni-muenster.de BACKGROUND: Neuroblastomas (NB) are a heterogeneous group of childhood tumours with a wide range of likelihood for tumour progression. As traditional parameters do not ensure completely accurate prognostic grouping, new molecular markers are needed for assessing the individual patient's prognosis more precisely. PATIENTS AND METHODS: 133 NB of all stages were analysed in blind-trial fashion for telomerase activity (TA), expression of surviving, and MYCN status. These data were correlated with other traditional prognostic indicators and disease outcome. RESULTS AND CONCLUSIONS: TA is a powerful independent prognostic marker for all stages and is capable of differentiating between good and poor outcome in putative "favourable" clinical or biological subgroups of NB patients. High surviving expression is associated with an adverse outcome, but is more difficult to interprete than TA because survivin expression needs to be accurately quantified to be of predictive value. We propose an extended progression model for NB including emerging prognostic markers, with emphasis on telomerase activity. PMID: 11528552 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 82: J Mol Med. 2001 Aug;79(8):428-36. Aggressive childhood neuroblastomas do not express caspase-8: an important component of programmed cell death. Teitz T, Lahti JM, Kidd VJ. Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA. tal.teitz@stjude.org Neuroblastomas that overexpress N-Myc due to amplification of the MYCN oncogene are aggressive tumors that become very resistant to treatment by chemotherapy and irradiation. to identify tumor suppressor genes in this group of neuroblastomas we analyzed the expression and function of both apoptosis-related cell cycle regulatory genes in cell lines and patient tumor samples. We found that in a high percentage of neuroblastoma cell lines and patient samples with amplified MYCN, caspase-8 mRNA is not expressed. The caspase-8 gene, CASP8, was deleted or silenced by methylation in the neuroblastoma cell lines while methylation of its promoter region was the predominant mechanism for its inactivation in the patient tumor samples. Reintroduction of caspase-8 into the neuroblastoma cell lines resensitized these cells to drug-induced and survival factor dependent apoptosis. Subsequently others have also shown that caspase-8 is silenced by methylation in neuroblastoma and peripheral neural ectodermal tumors, and that the caspase-9 regulator Apaf-1 is silenced by methylation in melanoma cell lines and patient samples. We conclude that caspase-8 acts as a tumor suppressor gene in neuroblastomas, that its silencing provides a permissive environment for MYCN gene amplification once the tumors are treated with chemotherapeutic drugs/irradiation, and that expression of this gene in these tumor cells may be of clinical benefit. We also discuss the possible significance of the neural crest cell progenitor cell origin and the silencing of important apoptotic regulators via methylation in both neuroblastoma and melanoma tumors. Publication Types: Review Review, Tutorial PMID: 11511973 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 83: Br J Cancer. 2001 Aug 17;85(4):531-7. Neuroblastomas with chromosome 11q loss and single copy MYCN comprise a biologically distinct group of tumours with adverse prognosis. Luttikhuis ME, Powell JE, Rees SA, Genus T, Chughtai S, Ramani P, Mann JR, McConville CM. Division of Medical and Molecular Genetics, University of Birmingham B15 2TT, UK. Neuroblastoma is a heterogeneous tumour and its effective clinical management is dependent on accurate prognostic evaluation. In approximately 25% of patients amplification of the MYCN oncogene is known to be associated with a poor outcome. In order to identify additional molecular markers with prognostic potential in non-MYCN-amplified neuroblastomas, we looked for a correlation between clinical outcome and loss of heterozygosity (LOH) on four chromosomes that frequently show alteration in neuroblastoma (chromosomes 3, 4, 11 and 14). Chromosome 11q loss (with frequent parallel loss of chromosomes 3p, 4p and/or 14q) was found exclusively in tumours without MYCN amplification and was significantly associated with poor event-free survival. The 2-year event-free survival rate for 11q LOH cases was 30%, compared to 34% for MYCN-amplified cases and 100% for cases without these abnormalities. While 11q LOH was associated predominantly with advanced-stage disease, 2 cases with low-stage disease and 11q LOH both suffered relapses. We conclude that chromosome 11q loss defines a biologically distinct group of tumours without MYCN amplification that appear to have potential for aggressive metastatic growth. Thus this genetic alteration may be an important new prognostic marker in neuroblastoma. Copyright 2001 Cancer Research Campaign. PMID: 11506492 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 84: Med Pediatr Oncol. 2001 Jan;36(1):83-92. Prognostic significance of DNA di-tetraploidy in neuroblastoma. Ladenstein R, Ambros IM, Potschger U, Amann G, Urban C, Fink FM, Schmitt K, Jones R, Slociak M, Schilling F, Ritter J, Berthold F, Gadner H, Ambros PF. Department of Paediatric Haemato-Oncology, St. Anna Children's Hospital, Vienna, Austria. ladenstein@ccri.univie.ac.at BACKGROUND: Identification of biological factors may provide tools to discriminate poor risk neuroblastoma patients of diagnosis, to ultimately offer risk adapted treatment intensity. PROCEDURES: Tumour cell DNA content, MYCN amplification (NMA), deletion of the short arm of chromosome 1 (del 1p) as well as three serological markers were assessed in 179 children with neuroblastoma. RESULTS: Localised regional disease (stage 1 to 3) was diagnosed in 98 patients, and disseminated disease in 81 patients (65 with stage 4, 16 with stage 4s). Median age at diagnosis was 12 months and the median observation time 4 years. Sixty-seven of 179 patients had near di-tetraploid tumours (37%), with a significantly worse prognosis of 44% overall survival at 4 years in comparison with 88% in near triploid tumours (P < .001). The near di-tetraploid group showed a significant correlation with additional adverse biological factors (NMA, del 1p: P < 0.001), age over 1 year (P< 0.001), clinical stage 4 (P< 0.001), elevated ferritin (P = 0.023), and elevated LDH (P< 0.001). Multivariate analysis based on the overall (OS) and event free survival (EFS) estimations revealed that near di-tetraploidy was the most powerful biological factor, with a P-value of <0.001 for EFS and OS, followed by NMA (P = 0.015) for OS and del 1p (P= 0.047) for EFS. CONCLUSIONS: This analysis underlines the important influence of near di-tetraploidy on prognosis, and suggests that more efforts should be undertaken to implement this factor in future studies. Publication Types: Multicenter Study PMID: 11464912 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 85: Med Pediatr Oncol. 2001 Jan;36(1):80-2. Association among EPHB2, TrkA, and MYCN expression in low-stage neuroblastomas. Tang XX, Evans AE, Zhao H, Cnaan A, Brodeur GM, Ikegaki N. Division of Oncology, Children's Hospital of Philadelphia, Pennsylvania 19104-4318, USA. BACKGROUND: The EPH family is the largest subfamily of receptor protein-tyrosine kinases, consisting of EPHA and EPHB subgroups. Ligands of EPH family receptors are called ephrins, which include ephrin-A and ephrin-B subgroups. We recently found that transcripts encoding the EPHB subgroup (EPHB) and the ephrin-B subgroup (EFNB) were expressed together in neuroblastoma (NB) cell lines. PROCEDURE: In this study, we examined the expression of EPHB and EFNB transcripts in 24 NB specimens representing all clinical stages. We found that several EPHB and EFNB transcripts were expressed together in all NBs examined. RESULTS: Among the transcripts examined, EPHB6 expression was most significantly associated with low stage tumors (stages 1, 2, and 4S; P = 0.0048). TrkA expression was significantly correlated with EPHB6, EFNB2, and EFNB3 expression (P < 0.01 in each case). Taken together, these data indicate that the expression of EPHB6, EFNB2, and EFNB3 may serve as prognostic indicators of favorable NBs. In the low-stage NBs without MYCN amplification, EPHB2 expression was correlated both with MYCN expression and with TrkA expression (P < 0.01 in each case). Moreover, MYCN expression was correlated with TrkA expression (P < 0.01) in the low-stage NBs. CONCLUSIONS: This observation points to the possibility that MYCN expression might contribute to favorable outcome of low-stage NBs. PMID: 11464911 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 86: Med Pediatr Oncol. 2001 Jan;36(1):75-9. Two-dimensional DNA electrophoresis identifies novel CpG islands frequently coamplified with MYCN in neuroblastoma. Wimmer K, Zhu XX, Lamb BJ, Kuick R, Ambros P, Kovar H, Thoraval D, Elkahloun A, Meltzer P, Hanash SM. Department of Pediatrics, University of Michigan, Ann Arbor, USA. katharina.wimmer@unive.ac.at BACKGROUND: Amplification of the oncogene MYCN in neuroblastoma has been found to correlate with aggressive tumour growth and is used as a predictor of clinical outcome. The MYCN amplicon is known to involve coamplification of extensive DNA regions. Therefore it is possible that other genes are coamplified in this amplicon and that they may play a role in the poor outcome of MYCN amplified tumours. PROCEDURE: We have implemented an approach for the two-dimensional separation of human genomic restriction fragments to detect and isolate as yet unknown amplified sequences in the MYCN amplicon in neuroblastoma. Using this approach we have recently cloned a novel gene referred to as NAG that is frequently coamplified with MYCN in neuroblastoma. RESULTS AND CONCLUSIONS: We report here the identification and cloning of two additional CpG islands that are amplified in neuroblastoma. One contains a sequence that is identical to the first intron of DDX1. The other represents a novel CpG island that is associated with an as yet unidentified gene. We show that the novel CpG island is located in close proximity to the MYCN locus on chromosome 2 and is as frequently coamplified with MYCN in neuroblastoma as NAG and DDX1. PMID: 11464910 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 87: Med Pediatr Oncol. 2001 Jan;36(1):67-74. Biological characteristics of neuroblastoma with partial deletion in the short arm of chromosome 1. Hiyama E, Hiyama K, Ohtsu K, Yamaoka H, Fukuba I, Matsuura Y, Yokoyama T. Department of General Medicine, Hiroshima University School of Medicine, Japan. eiso@mcai.med.hiroshima-u.ac.jp BACKGROUND: Neuroblastoma shows remarkable heterogeneity, resulting in favorable and unfavorable outcomes. It is well known that almost all cases with MYCN amplification have a poor prognosis. We have previously reported that unfavorable tumors show high telomerase activity, whereas favorable tumors show low or nil activity. We also found that the unfavorable neuroblastoma often have a loss of heterozygosity (LOH) at the MYCL locus. PROCEDURE: To clarify the biological and clinical profiles of tumors with genetic abnormalities of the short arm of chromosome 1, we performed deletion mapping on 1p on 92 neuroblastoma tissues and corresponding noncancerous samples obtained from 92 cases for 24 micro- or minisatellite loci. RESULTS: LOH was detected in at least one locus of 1p in 43 (47%) cases. All samples were classified into four groups according to the deleted pattern: interstitial deletion (group I, n = 20), short terminal deletion (group ST, n = 6), large terminal deletion (group LT, n = 17), and without detectable deletion (group N, n = 49). All group I cases, whose SRO (shortest region of overlap) was at 1p36.1-2, survived disease free, and none of them showed MYCN amplification or high telomerase activity except for one case. On the other hand, in group LT cases, who showed a large terminal deletion from D1S162 (1p32-pter), including the SRO of group 1, only 5 out of 17 have survived disease free, and 13 showed MYCN amplification or high telomerase activity. The six group ST cases showed small terminal deletion from 1p36.3 with modest prognosis, similar to the group N. CONCLUSIONS: Thus, we propose three loci, 1p36.1-2, 1p32-34, and 1p36.3, as the candidate loci of neuroblastoma suppressor genes on chromosome 1p responsible for groups I, LT, and ST, respectively. Among them, the 1p32-34 locus may be associated with aggressiveness of tumor progression, possibly due to MYCN amplification and/or telomerase reactivation, while the remaining two loci may not. PMID: 11464909 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 88: Med Pediatr Oncol. 2001 Jan;36(1):52-5. Analysis of genomic imprinting at 1p35-36 in neuroblastoma. Hogarty MD, Maris JM, White PS, Guo C, Brodeur GM. Division of Oncology, Children's Hospital of Philadelphia, Pennsylvania 19104-4318, USA. hogarty@kermit.oncol.chop.edu BACKGROUND: Deletion of the distal short arm of chromosome 1 occurs in 25-35% of primary neuroblastomas, and a putative tumor suppressor gene has been mapped to a consensus region of deletion at 1p36.2-36.3. Indirect evidence suggests the presence of an imprinted neuroblastoma suppressor gene within this region, as well as an additional nonimprinted, proximal suppressor gene, inactivation of which correlates with MYCN amplification. PROCEDURE: To test this hypothesis, we performed 1p loss of heterozygosity (LOH) studies on a series of neuroblastomas for which parental DNA had been collected. PCR-formatted polymorphic markers were used to determine the size of the 1p deletion and the parental origin of the deleted 1p homologue. RESULTS: Twenty-six neuroblastomas with 1p LOH were evaluated. Twenty-four had MYCN amplification, and of these, 15 demonstrated loss of the paternally inherited 1p. Two neuroblastomas with a single copy of MYCN were evaluated and both had deletion of the paternally inherited 1p, with one case exhibiting a small terminal deletion. In addition, we have reviewed 49 previously reported neuroblastomas where 1p LOH data and the parental origin of the deleted lp homologue were available. CONCLUSIONS: Analyzed together, these 75 neuroblastomas demonstrate random deletion of parental 1p homologues (P = 0.30). Further, tumors with smaller deletions (breakpoints distal to D1S201 or D1S7) showed a random loss of the parental 1p homologues (P = 0.59), contrary to the expected preferential maternal 1p deletion if an imprinted suppressor gene mapped to this region. However, 19 tumors with 1p LOH and single copy MYCN had deletion of the maternal 1p homologue preferentially (P = 0.02), which does not exclude the possibility that loss of an imprinted suppressor gene plays a role in this subset. Publication Types: Review Review of Reported Cases PMID: 11464906 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 89: Med Pediatr Oncol. 2001 Jan;36(1):247-50. Localised and unresectable neuroblastoma in infants: excellent outcome with primary chemotherapy. Neuroblastoma Study Group, Societe Francaise d'Oncologie Pediatrique. Rubie H, Plantaz D, Coze C, Michon J, Frappaz D, Baranzelli MC, Chastagner P, Peyroulet MC, Hartmann O; Neuroblastoma Study Group, Societe Francaise d'Oncologie Pediatrique. Unite d'Hemato-Oncologie Pediatrique, Hjpital des Enfants, Toulouse, France. rubie.h@chu-toulouse.fr PROCEDURE: Infants with neuroblastoma (NB) were assessed according to INSS recommendations, including MIBG scan and extensive bone marrow staging to eliminate metastatic spread. Patients with unresectable tumour received chemotherapy, including two courses of carboplatin-etoposide (CE) and two of vincristinecyclophosphamide-doxorubicin (CAdO). Post-operative treatment was to be given only in infants with MYCN amplification. Between 1990 and 1994, 52 consecutive children were registered. RESULTS: Among the 44 patients who received CE as a first course, the response rate was (66%) and the primary could be removed in all children but one, who was in remission. The toxicity was mainly haematological and was always manageable. The 5 year overall survival (OS) and event-free survival (EFS) were 94 and 90 +/- 8%, respectively, with a median follow-up of 48 months. The outcome of infants with no MYCN amplification was excellent; OS and EFS were, respectively, 97 and 94%. CONCLUSIONS: Chemotherapy allows surgical excision and excellent outcome in infants with localised and unresectable NB. Less intensive Chemotherapy should be investigated in such patients. PMID: 11464897 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 90: Med Pediatr Oncol. 2001 Jan;36(1):24-7. Allelic deletion at chromosome bands 11q14-23 is common in neuroblastoma. Maris JM, Guo C, White PS, Hogarty MD, Thompson PM, Stram DO, Gerbing R, Matthay KK, Seeger RC, Brodeur GM. Division of Oncology, Children's Hospital of Philadelphia, Pennsylvania 19104-4138, USA. maris@email.chop.edu BACKGROUND: Neuroblastoma tumorigenesis may involve the differential inactivation of multiple tumor suppressor genes. Recent data have suggested that a neuroblastoma suppressor gene may be located on the long arm of chromosome 11 (11q). PROCEDURE: We therefore analyzed 295 primary neuroblastomas from a representative group of patients for loss of heterozygosity (LOH) at 25 polymorphic markers spanning 11q. RESULTS: LOH was observed in 129 primary neuroblastomas (44%), and a common region of LOH mapped to 11q14-23. No correlation was found between 11q LOH and adverse prognostic variables, but a strong inverse relationship between 11q LOH and MYCN amplification (P < 0.001) was observed. There was no difference in overall survival when patients were stratified by 11q LOH status. However, 11q LOH was associated with a decreased overall survival probability when patients whose tumors had a single copy of MYCN were analyzed separately (P = 0.008). CONCLUSION: These data support the hypothesis that a tumor suppressor gene mapping within 11q14-23 is frequently inactivated during the malignant evolution of neuroblastoma. Publication Types: Multicenter Study PMID: 11464895 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 91: Med Pediatr Oncol. 2001 Jan;36(1):227-30. N7: a novel multi-modality therapy of high risk neuroblastoma (NB) in children diagnosed over 1 year of age. Cheung NK, Kushner BH, LaQuaglia M, Kramer K, Gollamudi S, Heller G, Gerald W, Yeh S, Finn R, Larson SM, Wuest D, Byrnes M, Dantis E, Mora J, Cheung IY, Rosenfield N, Abramson S, O'Reilly RJ. Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. Cheungn@MSKCC.org BACKGROUND: The N7 protocol for poor-risk neuroblastoma uses dose-intensive chemotherapy (as in N6 protocol [Kushner et al.: J Clin Oncol 12:2607-2613, 1994] but with lower dosing of vincristine) for induction, surgical resection and 2100 cGy hyperfractionated radiotherapy for local control, and for consolidation, targeted radioimmunotherapy with 131I-labeled anti-GD2 3F8 monoclonal antibody and immunotherapy with unlabeled/unmodified 3F8 (400 mg/m2). PROCEDURE: The chemotherapy consists of: cyclophosphamide 70 mg/kg/d x 2 and a 72-hr infusion of doxorubicin 75 mg/m2 plus vincristine 2 mg/m2, for courses 1, 2, 4, and 6; and cisplatin 50 mg/m2/d x 4 and etoposide 200 mg/m2/d x 3, for courses 3, 5, and 7. 131I-3F8 is dosed at 20 mCi/kg, which is myeloablative and therefore necessitates stem-cell support. RESULTS: Of the first 24 consecutive previously untreated patients more than 1 year old at diagnosis, 22 were stage 4 and two were unresectable stage 3 with MYCN amplification. Chemotherapy achieved CR/VGPR in 21 of 24 patients. Twenty patients to date have completed treatment with 131I-3F8, and 15 patients have completed all treatment. With a median follow-up of 19 months, 18 of 24 patients remain progression-free. CONCLUSIONS: Major toxicities were grade 4 myelosuppression and mucositis during chemotherapy, and self-limited pain and urticaria during antibody treatment. Late effects include hearing deficits and hypothyroidism. Publication Types: Clinical Trial PMID: 11464891 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 92: Med Pediatr Oncol. 2001 Jan;36(1):224-6. Somatostatin receptor type 2 gene expression in neuroblastoma, measured by competitive RT-PCR, is related to patient survival and to somatostatin receptor imaging by indium -111-pentetreotide. Orlando C, Raggi CC, Bagnoni L, Sestini R, Briganti V, La Cava G, Bernini G, Tonini GP, Pazzagli M, Serio M, Maggi M. Dep of Clinical Physiopathology, University of Florence, Italy. c.orlando@dfc.unifi.it BACKGROUND: We previously reported that human neuroblastoma cell lines and primary neuroblastoma tumors expressed a variable amount of mRNA for type 2 somatostatin (sst2) receptor gene. We also found that high level of sst2 expression were positively related to patient survival. PROCEDURE: We studied retrospectively 49 primary neuroblastomas. To detect and measure sst2 mRNA expression we developed a quantitative RT-PCR based on competitive PCR. When possible the number of MYCN copies was also measured with competitive PCR. RESULTS;. We found that the lowest level of sst2 mRNA was detected in advanced stages of neuroblastomas (stage IV) when compared with the other stages (P< 0.005). Patients with high levels of sst2 expression (>7 x 10(7) molecules/microg RNA) had a cumulative survival better than those with low sst2 expression (P < 0.0005). This predictive independent value of sst2 (P= 0.005) is retained after stratification for N-myc amplification. Finally we verified that the ex vivo sst2 gene expression in tumor samples was positively related (P < 0.01) to the in vivo semiquantitative determination of sst2 protein, assessed by 111In-pentetreotide imaging. CONCLUSIONS: Our data indicate that the measurement of sst2 mRNA measurement could represent a relevant tool in the prediction of neuroblastoma outcome, independently from MYCN amplification. Publication Types: Evaluation Studies PMID: 11464890 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 93: Med Pediatr Oncol. 2001 Jan;36(1):169-76. Relationship between histopathological features, MYCN amplification, and prognosis: a UKCCSG study. United Kingdom Children Cancer Study Group. George RE, Variend S, Cullinane C, Cotterill SJ, McGuckin AG, Ellershaw C, Lunec J, Pearson AD; United Kingdom Children Cancer Study Group. Cancer Research Unit, Medical School, University of Newcastle upon Tyne, United Kingdom. Histological sections from 231 patients with neuroblastoma were reviewed and morphological features and their relationship to age, stage, MYCN amplification (in 128 tumours by Southern analyses), and clinical outcome (based on Shimada risk grouping) determined. Stage 4 disease was associated with poorly differentiated and undifferentiated tumours (P = 0.001), an MKI of >2% (P< 0.001), and Shimada unfavourable histology (UHi) P< 0.0001. In univariate analysis MKI was significant in predicting a poorer relapse-free survival (RFS), low vs. intermediate and high (P< 0.001). Age, MYCN amplification, and Shimada UH also emerged as significant variables. There was a higher proportion of MYCN-amplified tumours with Shimada UH (P = 0.03), and this group had a decreased RFS (P = 0.002). In patients with Shimada FH, MYCN amplification did not significantly predict a poor prognosis. In those with stage 4 disease, Shimada classification was not significant in predicting survival (P = 0.97); the same was true for those over the age of 1 year (P = 0.66). In multivariate analysis, MYCN amplification and Shimada UH both emerged as independent prognostic factors. In conclusion, morphological features assigned some subsets of patients to prognostic risk groups. Most MYCN-amplified tumours have unfavourable histology and a poorer prognosis. However, in patients with stage 4 disease and those over the age of 1 year, other factors that may influence prognosis should be determined. Publication Types: Review Review, Multicase PMID: 11464876 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 94: Med Pediatr Oncol. 2001 Jan;36(1):157-9. Biological aspects of neuroblastomas identified by mass screening in Quebec. Brodeur GM, Look AT, Shimada H, Hamilton VM, Maris JM, Hann HW, Leclerc JM, Bernstein M, Brisson LC, Brossard J, Lemieux B, Tuchman M, Woods WG. Division of Oncology, Children's Hospital of Philadelphia, PA 19104-4318, USA. brodeur@email.chop.edu BACKGROUND: Neuroblastoma has several characteristics that suggest that preclinical diagnosis might improve outcome. Therefore, the Quebec Neuroblastoma Screening Project was undertaken from 1989 to 1994 to examine infants at 3 weeks and 6 months by measuring urinary catecholamine metabolites. PROCEDURE: Over the 5-yr period, 45 tumors were detected by screening, 20 were identified clinically prior to the third week, and 64 were identified clinically at a later time. We analyzed available tumors for Shimada histopathology, tumor ploidy, MYCN copy number and serum ferritin. RESULTS: Of the tumors detected by screening, only 2 of 45 tested had unfavorable histology, 2 of 45 had diploid or tetraploid DNA content, 0 of 43 had MYCN amplification, and 4 of 44 had elevated serum ferritin. All of these patients are alive and well. The 20 patients detected prior to the 3-week screen had similar biological characteristics. In contrast, of the patients detected clinically after 3 weeks of age, 19 of 51 testedhad unfavorable histology, 25 of 66 had diploid or tetraploid tumors, 12 of 56 had MYCN amplification, and 14 of 54 had elevated ferritin. CONCLUSIONS: The difference between the screened and clinically detected cases was highly significant for each biological variable. Preliminary data on other biological variables, such as neurotrophin expression and allelic loss on 1 p in these patients are consistent with the above findings. These data suggest that mass screening for neuroblastoma at or before 6 months of age detects almost exclusively tumors that have favorable biological characteristics, many of which might have regressed spontaneously. Thus, continued mass screening for neuroblastoma at 6 months is unlikely to accomplish its intended goal, and should probably be discontinued. PMID: 11464873 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 95: Med Pediatr Oncol. 2001 Jan;36(1):14-9. 17q gain in neuroblastoma predicts adverse clinical outcome. U.K. Cancer Cytogenetics Group and the U.K. Children's Cancer Study Group. Bown N, Lastowska M, Cotterill S, O'Neill S, Ellershaw C, Roberts P, Lewis I, Pearson AD; U.K. Cancer Cytogenetics Group and the U.K. Children's Cancer Study Group. Department of Human Genetics, University of Newcastle upon Tyne, United Kingdom. Nicholas.bown@ncl.ac.uk BACKGROUND: It is now recognized that gain of chromosome 17 material is the most frequent genetic abnormality of neuroblastoma cells. Several studies have linked 17q gain with known adverse prognostic factors: patient age >1 year, advanced stage disease, deletion of chromosome arm 1 p, and amplification of the MYCN oncogene. We sought to further investigate the clinical and prognostic associations of chromosome 17 status in relation to other well-established predictive factors. PROCEDURE: In a collaborative study by UK cytogenetics centres, we compiled a series of 104 neuroblastoma tumours for which the status of chromosome 17 was confidently defined by cytogenetics, metaphase or interphase FISH, or CGH analysis. The results were correlated with data on 1p and MYCN, and with centrally collated clinical and survival information. RESULTS: Gain of 17q (i.e., unbalanced gain of segment 17q21-qter) was found in 66.3% of tumours, while 33.7% showed a '17q normal' status (i.e., no gain at all, or gain of whole chromosome 17 relative to ploidy). Gain of 17q was strongly associated with advanced stage disease, patient age >1 year, 1p deletion, and MYCN amplification (all P< 0.01). In univariate analysis, 17q gain was a significant predictor of adverse outcome (projected 5 year relapse-free survival 15.6% compared to 75.2% in cases lacking this feature in tumour cells; (P < 0.0001). In multivariate analysis, 17q gain was more strongly associated with adverse outcome than was either stage (Stage 4 vs other combined) or 1p status. CONCLUSION: We conclude that gain of chromosome segment 17q21-qter is of great biological and clinical importance in neuroblastoma, and that its detection at diagnosis should be a priority. Publication Types: Multicenter Study PMID: 11464868 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 96: Med Pediatr Oncol. 2001 Jan;36(1):111-4. Expression of Fas (APO-1/CD95) and Fas ligand (FasL) in human neuroblastoma. Gross N, Balmas K, Beretta Brognara C, Tschopp J. Onco-hematology Unit, University Hospital, Lausanne, Switzerland. Nicole.Gross@chuv.hospvd.ch BACKGROUND AND PROCEDURE: To determine the possible role of Fas/FasL system in the particularly heterogeneous behaviour of neuroblastoma (NB), we have measured the functional expression of Fas and its ligand, FasL, in primary neuroblastoma samples and cell lines by immunohistochemistry and flow cytometry. RESULTS: Our results reveal that while Fas expression is associated with low stage and more mature tumors, heterogeneous FasL expression was mostly detected in high stage tumors, with our apparent correlation to MYCN amplification. Flow cytometric analysis of cell lines demonstrated a high expression of Fas in epithelial-type, HLA class I positive cell lines, which was lost upon activation with phorbol esters. In contrast, Fas ligand was detected in only a small subset of cell lines. CONCLUSIONS: In some cell lines, cytotoxic assays revealed the ability of NB-associated Fas receptor to transduce an apoptotic signal upon triggering. The pattern of functional Fas/FasL expression in tumours and cell lines suggests that this system may be involved in the evasion of highly malignant neuroblastoma cells to host immune response. PMID: 11464860 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 97: Med Pediatr Oncol. 2001 Jan;36(1):11-3. Distal chromosome 17 gains in neuroblastomas detected by comparative genomic hybridization (CGH) are associated with a poor clinical outcome. Brinkschmidt C, Christiansen H, Terpe HJ, Simon R, Lampert F, Boecker W, Dockhorn-Dworniczak B. Department of Pathology, University of Munster, Germany. brinkch@uni-muenster.de PROCEDURE: To establish the significance of chromosome 17 aberrations in the biology of neuroblastomas, the fresh-frozen material of 53 primary neuroblastomas (average patient age: 20.8 months; stage 1 or 2: n = 10; stage 3: n = 10; stage 4: n = 10; stage 4s: n = 23) was studied by means of comparative genomic hybridization (CGH). Follow-up data were available for 52 of 53 cases studied (average follow-up period: 26.4 months). Except for one, all cases had previously been analyzed for MYCN status (semiquantitative Southern blot analysis). Studies of LOH 1p36 (VNTR-PCR) had been performed on 28 of 53 cases. RESULTS: Chromosome 17 gains were detected in 46 of 53 (86.8%) cases. Whole chromosome gains were mostly restricted to localized tumors (stage 1 or 2: 9 of 10 cases; stage 4s:19 of 23; stage 3: 2 of 10; stage 4:0 of 10 cases), whereas distal 17 gains were significantly associated with clinically advanced tumor stages and patients aged over 1 year at diagnosis. Univariate analyses revealed a statistically significant correlation of distal 17q gains with overall survival (P< 0.01, MYCN amplification: P< 0.01; 1p deletion: P< 0.01) and an elevated recurrency rate (17q: P= 0.02, MYCN amplification: P = 0.05; 1p deletion P= 0.3). There was a strong coincidence of distal 17q gains and 1p deletion or MYCN amplification (P < 0.01). CONCLUSION: Our data indicate that distal chromosome 17q gains are of major prognostic relevance for neuroblastoma patients. However, studies on a larger series of tumors have to be performed to assess whether or not these alterations are independent prognostic markers of a poor clinical outcome. PMID: 11464859 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 98: Med Pediatr Oncol. 2001 Jan;36(1):1-4. Intratumoural heterogeneity of 1p deletions and MYCN amplification in neuroblastomas. Ambros PF, Ambros IM, Kerbl R, Luegmayr A, Rumpler S, Ladenstein R, Amann G, Kovar H, Horcher E, De Bernardi B, Michon J, Gadner H. CCRI, Children's Cancer Research Institute, St Anna Kinderspital, Vienna, Austria. ambros@ccri.univie.ac.at BACKGROUND: At least three genetic hallmarks identify aggressive tumour behaviour in neuroblastomas; amplification of the oncogene MYCN; deletion (loss of heterozygosity [LOH]) at the short arm of chromosome 1 (del1p36), seen in approximately 28% of the cases; and di-tetraploidy. The MYCN oncogene is amplified in approximately 23% of all neuroblastomas and becomes important for the stratification of therapy in localised and 4s tumours. Up to now, it has been believed that the genetic constellation of neuroblastic tumours is stable and does not alter during tumour evolution or during tumour progression. PROCEDURE: Using fluorescence in situ hybridisation techniques (FISH) to investigate different tumour areas on touch preparations and histological sections, we show that genetic heterogeneity can be detected in neuroblastomas, especially in tumours detected by urinary mass screening. CONCLUSION: The identification of such cell clones is important, because the MYCN amplification and/or the deletion at 1p36 appear to be responsible for aggressive local growth and development of metastases. PMID: 11464855 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 99: Genes Chromosomes Cancer. 2001 Aug;31(4):345-56. Amplification of Mycn, Ddx1, Rrm2, and Odc1 in rat uterine endometrial carcinomas. Karlsson A, Helou K, Walentinsson A, Hedrich HJ, Szpirer C, Levan G. Department of Cell and Molecular Biology-Genetics, Goteborg University, SE 405 30 Gothenburg, Sweden. The BDII rat is genetically predisposed to estrogen-dependent endometrial adenocarcinoma and represents a valuable model for this type of tumor. Tumors arising in strain crosses involving the BDII rats had previously been screened for DNA copy number changes using comparative genome hybridization (CGH). It was found that extra copies of the proximal region of rat chromosome (RNO) 6 commonly could be detected in these tumors. Based on RH-mapping data and comparative mapping with mouse and human, seven cancer-related genes were predicted to be situated in RNO6q14-q16. Rat PACs were isolated for the N-myc proto-oncogene (Mycn), apolipoprotein B (Apob), the DEAD box gene 1 (Ddx1), ornithine decarboxylase 1 (Odc1), proopiomelanocortin (Pomc1), ribonucleotide reductase, M2 polypeptide (Rrm2), and syndecan 1 (Sdc1). The localization of the genes to the region was verified by FISH (fluorescence in situ hybridization) mapping, and the detailed order among them was determined by dual-color FISH. By Southern blot analysis, it was found that the Mycn locus was highly amplified in two out of 10 cell cultures derived from the tumors. In one of them (designated RUT30), the amplification level of Mycn was estimated at 140x. Two other genes were coamplified (Ddx1 and Rrm2) at much lower levels. Similarly, in another culture (designated RUT2), Mycn was amplified more than 40x, whereas three of the other genes (Ddx1, Rrm2, and Odc1) were coamplified at lower levels. Using FISH on metaphase chromosomes from the cell cultures analyzed, the amplified sequences were shown to be located in typical HSRs. With competitive RT-PCR, distinct overexpression of Mycn and Ddx1 could be demonstrated in both RUT2 and RUT30. In addition, Mycn was overexpressed in two other tumors not exhibiting Mycn amplification. Taken together, our results suggest that overexpression of Mycn plays an important role in the development of endometrial cancer in the BDII rat. In humans, Mycn amplification has been reported mainly from tumors of neuronal origin. To our knowledge, this is the first report of Mycn amplification and overexpression in hormone-dependent tumors. Copyright 2001 Wiley-Liss, Inc. PMID: 11433525 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 100: Neoplasia. 2001 Mar-Apr;3(2):105-9. Detection of MYCN gene amplification in neuroblastoma by fluorescence in situ hybridization: a pediatric oncology group study. Mathew P, Valentine MB, Bowman LC, Rowe ST, Nash MB, Valentine VA, Cohn SL, Castleberry RP, Brodeur GM, Look AT. Department of Experimental Oncology, St. Jude Children's Research Hospital, Memphis, TN, USA. To assess the utility of fluorescence in situ hybridization (FISH) for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (< or =30% replacement) in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker. PMID: 11420745 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 101: J Clin Oncol. 2001 Jun 15;19(12):3080-90. Comprehensive genetic and histopathologic study reveals three types of neuroblastoma tumors. Lastowska M, Cullinane C, Variend S, Cotterill S, Bown N, O'Neill S, Mazzocco K, Roberts P, Nicholson J, Ellershaw C, Pearson AD, Jackson MS; United Kingdom Children Cancer Study Group and the United Kingdom Cancer Cytogenetics Group. Human Genetics Unit, School of Biochemistry and Genetics, University of Newcastle upon Tyne, United Kingdom. M.A.Lastowska@ncl.ac.uk PURPOSE: To determine the relationship between multiple genetic features, tumor morphology, and prognosis in neuroblastoma. PATIENTS AND METHODS: The genetic alterations and morphologic features that underpin three histopathologic risk classifications were analyzed in 108 neuroblastoma patients. Tumors were subdivided into four groups based on the three most frequent and prognostically significant genetic alterations (17q gain, 1p deletion, and MYCN amplification), and all other genetic, morphologic, and clinical data were analyzed with respect to these groups. RESULTS: Our analyses identify three nonoverlapping tumor types with distinct genetic and morphologic features, defined here as types 1, 2, and 3. Type 1 tumors show none of the three significant genetic alterations and have good prognosis. Both type 2 (17q gain only or 17q gain and 1p del) and type 3 (17q gain, 1p del, and MYCN amplification) tumors progress. However, these tumor types are distinguished clinically by having significantly different median age at diagnosis and median progression-free survival (PFS). Multivariate analysis indicates that 17q gain is the only independent prognostic factor among all genetic, histopathologic, and clinical factors analyzed. Among histopathologic risk systems, the International Neuroblastoma Pathology Classification was the best predictor of PFS. CONCLUSION: Our results indicate that specific combinations of genetic changes in neuroblastoma tumors contribute to distinct morphologic and clinical features. Furthermore, the identification of two genetically and morphologically distinct types of progressing tumors suggests that possibilities for different therapeutic regimens should be investigated. PMID: 11408505 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 102: Am J Pathol. 2001 Jun;158(6):2067-77. p53 cellular localization and function in neuroblastoma: evidence for defective G(1) arrest despite WAF1 induction in MYCN-amplified cells. Tweddle DA, Malcolm AJ, Cole M, Pearson AD, Lunec J. Cancer Research Unit, The Medical School, University of Newcastle, Newcastle-upon-Tyne, United Kingdom. d.a.tweedle@newcastle.ac.uk This study investigated the hypothesis that p53 accumulation in neuroblastoma, in the absence of mutation, is associated with functional inactivation, which interferes with downstream mediators of p53 function. To test this hypothesis, p53 expression, location, and functional integrity was examined in neuroblastoma by irradiating 6 neuroblastoma cell lines and studying the effects on p53 transcriptional function, cell cycle arrest, and induction of apoptosis, together with the transcriptional function of p53 after irradiation in three ex vivo primary, untreated neuroblastoma tumors. p53 sequencing showed five neuroblastoma cell lines, two of which were MYCN-amplified, and that all of the tumors were wild-type for p53. p53 was found to be predominantly nuclear before and after irradiation and to up-regulate the p53 responsive genes WAF1 and MDM2 in wild-type p53 cell lines and a poorly-differentiated neuroblastoma, but not a differentiating neuroblastoma or the ganglioneuroblastoma part of a nodular ganglioneuroblastoma in short term culture. This suggests intact p53 transcriptional activity in proliferating neuroblastoma. Irradiation of wild-type p53 neuroblastoma cell lines led to G(1) cell cycle arrest in cell lines without MYCN amplification, but not in those with MYCN amplification, despite induction of WAF1. This suggests MYCN amplification may alter downstream mediators of p53 function in neuroblastoma. PMID: 11395384 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 103: J Clin Oncol. 2001 Jun 1;19(11):2821-8. Hyperfractionated low-dose radiotherapy for high-risk neuroblastoma after intensive chemotherapy and surgery. Kushner BH, Wolden S, LaQuaglia MP, Kramer K, Verbel D, Heller G, Cheung NK. Departments of Epidemiology and Biostatistics, Pediatrics, and Radiation Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY. PURPOSE: To assess prognostic factors for local control in high-risk neuroblastoma patients treated with hyperfractionated 21-Gy total dose to consolidate remission achieved by dose-intensive chemotherapy and surgery. PATIENTS AND METHODS: Patients with high-risk neuroblastoma in first remission received local radiotherapy (RT) totaling 21 Gy in twice-daily 1.5-Gy fractions. RT to the primary site followed dose-intensive chemotherapy and tumor resection; the target field encompassed the extent of tumor at diagnosis, plus 3-cm margins and regional lymph nodes. RT to distant sites followed radiologic evidence of response. Local failure was correlated with clinical factors (including other consolidative treatments) and biologic findings. RESULTS: Of 99 consecutively irradiated patients followed for a median of 21.1 months from RT, 10 relapsed in or at margins of RT fields at 1 to 27 months (median, 14 months). At 36 months after RT, the probability of primary-site failure was 10.1% +/- 5.3%. No primary-site relapses occurred among the 23 patients whose tumors were excised at diagnosis, but there were three such relapses among the seven patients who were irradiated with evidence of residual disease in the primary site. Four of 18 patients with MYCN-amplified disease and serum lactate dehydrogenase greater than 1,500 U/L had local failures (23.4% +/- 10.7% risk at 18 months). Acute radiotoxicities were insignificant, but three of 35 patients followed for > or = 36 months had short stature from decreased growth of irradiated vertebra. CONCLUSION: Hyperfractionated 21-Gy RT is well tolerated and, together with dose-intensive chemotherapy and surgery, may help in local control of high-risk neuroblastoma. Extending the RT field to definitively encompass regional nodal groups may improve results. Visible residual disease may warrant higher RT dosing. Patients with biologically unfavorable disease may be at increased risk for local failure. RT to the primary site may not be necessary when tumors are excised at diagnosis. Publication Types: Clinical Trial PMID: 11387353 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 104: Diagn Mol Pathol. 2001 Jun;10(2):100-4. Rearrangement in the coding region of the MYCN gene in a subset of amplicons in a case of neuroblastoma with MYCN amplification. Chen B, Jhanwar SC, Ladanyi M. Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. chenb@mskcc.org The MYCN gene is often amplified but rarely rearranged in neuroblastoma. We report, for the first time, a rearrangement within the MYCN coding region in a metastatic neuroblastoma in a 3-year-old boy with MYCN amplification in his primary tumor. The rearrangement occurred 46 nucleotides downstream from the ATG codon in exon 2 of MYCN. The amplification level of the rearranged copies of the MYCN gene was lower than that of the unrearranged copies of MYCN. These results indicate that the rearrangement occurred after initial MYCN gene amplification. Monochromosomal somatic cell hybrid mapping of the novel region fused to exon 2 of MYCN localized it to chromosome 2, suggesting that this rearrangement resulted from an interstitial deletion, presumably within the MYCN amplicon itself. Publication Types: Case Reports PMID: 11385318 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 105: Lab Invest. 2001 May;81(5):717-23. Detection of multiple gene amplifications in glioblastoma multiforme using array-based comparative genomic hybridization. Hui AB, Lo KW, Yin XL, Poon WS, Ng HK. Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China. We have used a new method of genomic microarray to investigate amplification of oncogenes throughout the genome of glioblastoma multiforme (GBM). Array-based comparative genomic hybridization (array CGH) allows for simultaneous examination of 58 oncogenes/amplicons that are commonly amplified in various human cancers. Amplification of multiple oncogenes in human cancers can be rapidly determined in a single experiment. Tumor DNA and normal control DNA were labeled by nick translation with green- and red-tagged nucleotides, respectively. Instead of hybridizing to normal metaphase chromosomes in conventional comparative genomic hybridization (CGH), the probes of the mixed fluorescent labeled DNA were applied to genomic array templates comprised of P1, PAC, and BAC clones of 58 target oncogenes. The baseline for measuring deviations was established by performing a series of independent array CGH using test and reference DNA made from normal individuals. In the present study, we examined fourteen GBMs (seven cell lines and seven tumours) with CGH and array CGH to reveal the particular oncogenes associated with this cancer. High-level amplifications were identified on the oncogenes/amplicons CDK4, GLI, MYCN, MYC, MDM2, and PDGFRA. The highest frequencies of gains were detected on PIK3CA (64.3%), EGFR (57.1%), CSE1L (57.1%), NRAS (50%), MYCN (42.9%), FGR (35.7%), ESR (35.7%), PGY1 (35.7%), and D17S167 (35.7%). These genes are suggested to be involved in the GBM tumorigenesis. PMID: 11351043 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 106: Am J Pathol. 2001 May;158(5):1579-84. In vivo elimination of acentric double minutes containing amplified MYCN from neuroblastoma tumor cells through the formation of micronuclei. Valent A, Benard J, Clausse B, Barrois M, Valteau-Couanet D, Terrier-Lacombe MJ, Spengler B, Bernheim A. Laboratoire de Genomique Cellulaire des Cancers Centre National de la Recherche Scientifique Unite Mixte de Recherche, Villejuif, France. avalent@igr.fr Neuroblastoma, the most common solid extracranial neoplasm in children, shows an appreciable variability in clinical evolution. Amplification of the MYCN oncogene in this tumor is detected in 25 to 30% of cases and is associated with poor clinical outcome. In this study, quantitative polymerase chain reaction and fluorescence in situ hybridization were used to determine MYCN amplification status in 46 neuroblastoma tumors. MYCN amplification was detected in tumors from 11 patients. Fluorescence in situ hybridization revealed the presence of micronuclei containing amplified MYCN sequences in 8 of the 11 tumors. Micronuclei are indicative of spontaneous elimination or loss of amplified sequences by tumor cells. Because the elimination of amplified sequences can be enhanced in vitro by specific drugs such as hydroxyurea, our observations suggest a new therapeutic strategy specifically targeted to cells with amplified genes. PMID: 11337354 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 107: Genes Chromosomes Cancer. 2001 Jun;31(2):172-81. Three chromosomal rearrangements in neuroblastoma cluster within a 300-kb region on 1p36.1. Spieker N, Beitsma M, Van Sluis P, Chan A, Caron H, Versteeg R. Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, Netherlands. Deletions in the short arm of chromosome 1 (1p36) and MYCN amplification are common in neuroblastoma. Previously we showed evidence of at least two different neuroblastoma tumor-suppressor loci on 1p. One is associated with MYCN single-copy tumors and maps distal on 1p36.3. A second, more proximal locus maps to 1p36.1 and is deleted in about 90% of neuroblastomas with MYCN amplification. The cell line UHG-NP has the smallest 1p36 deletion of all neuroblastoma cell lines with MYCN amplifications. We assume that the more proximal locus maps within this deletion, close to its proximal border. Here we present the exact localization of the 1p deletion breakpoint of UHG-NP. A 600-kb PAC contig spanning the breakpoint was analyzed for genes and aberrations. Two more neuroblastoma-associated aberrations were mapped within 150 kb of the UHG-NP breakpoint. Within the contig, we identified nine genes expressed in neuroblastoma cells. One of these genes, AML2, maps 200 kb distal to the UHG-NP breakpoint but is expressed only rarely in neuroblastoma and showed no mutations. Copyright 2001 Wiley-Liss, Inc. PMID: 11319804 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 108: Int J Cancer. 2001 May 20;95(3):176-83. Expression of P27(KIP1) is prognostic and independent of MYCN amplification in human neuroblastoma. Bergmann E, Wanzel M, Weber A, Shin I, Christiansen H, Eilers M. Universitats-Kinderklinik, Marburg, Germany. Amplification of the MYCN gene is significantly associated with an unfavorable prognosis and rapid progression in human neuroblastoma tumors. One potential mechanism by which MYCN may cause these effects is by deregulating cell proliferation. Tissue culture experiments support a model in which MYC genes stimulate cell cycle progression by antagonizing the function of the cell cycle inhibitor p27(kip1). In culture, activation of MYC induces both sequestration of p27(kip1) by cyclin D complexes and its subsequent proteolytic degradation. We have tested whether this model applies to human neuroblastoma in a retrospective study of 100 primary tumor biopsy samples from neuroblastoma patients with a documented follow-up. Consistent with this hypothesis, MYCN-amplified tumors express high levels of both cyclin A and proliferating cell nuclear antigen, 2 marker proteins of cell proliferation. Further, expression levels of p27(kip1) are of prognostic significance in human neuroblastoma patients. Similar to tissue culture systems, p27(kip1) is sequestered by cyclin D complexes in a subset of human neuroblastoma samples. Surprisingly, however, expression levels of p27(kip1) are prognostic independent of MYCN amplification, and tumors that have an amplified MYCN gene do not express elevated levels of D-type cyclins or contain significantly lower levels of p27(kip1). Our data do not support a model in which regulation of p27(kip1) function is an important mechanism by which amplified MYCN deregulates cell proliferation in neuroblastoma. Copyright 2001 Wiley-Liss, Inc. PMID: 11307151 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 109: Pathology. 2001 Feb;33(1):108-11. Solid variant of alveolar rhabdomyosarcoma with unbalanced t(2;13) and hypotetraploidy, without MYCN amplification. Smith A, Sharma P, Tomlinson J, Robson L, Goldrick A. Department of Cytogenetics, Royal Alexandra Hospital for Children, Westmead, NSW, Australia. The histological subtype of alveolar rhabdomyosarcoma (AR) is characterised by the cytogenetic translocation t(2;13)(q35;q14) in approximately 70% of cases, a rearrangement rarely present in the embryonal rhabdomyosarcoma (ER) subtype. The MYCN gene is amplified in some cases of AR. We present a young man with an unusual pattern, namely solid variant of AR with hypotetraploidy and the t(2;13) in an unbalanced form. The MYCN gene was not amplified on FISH, but showed increased copy number, consistent with ploidy. Publication Types: Case Reports PMID: 11280599 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 110: Pediatr Dev Pathol. 2001 Jan-Feb;4(1):62-7. Telomerase activity as a prognostic factor in neuroblastomas. Streutker CJ, Thorner P, Fabricius N, Weitzman S, Zielenska M. Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada. Neuroblastoma is the most common extracranial solid tumor of early childhood. This tumor demonstrates significant heterogeneity with respect to pathologic, genetic, and clinical features. The outcome varies from spontaneous regression or maturation to rapid progression, despite aggressive therapy. Prognostic factors have been found that identify those tumors which have a high probability of aggressive behavior; these factors include unfavorable histology, MYCN copy number, deletions of the short arm of chromosome 1, DNA content, and TRK-A (high-affinity receptor protein for nerve growth factor) expression. Recent studies have suggested that high levels of telomerase activity also correlate with poor clinical outcome. We investigated this relationship in 40 patients with untreated neuroblastoma, using a PCR-ELISA assay for telomerase activity. In these patients, 23 tumors had no or minimal telomerase activity whereas 15 had high levels of activity. In two tumors, telomerase activity was not assessable. There was significant correlation between the telomerase activity and MYCN copy number, 1p deletions, and TRK-A expression, as well as patient age, clinical stage, and outcome. The histological classification of the tumors was not significantly different between the two groups, being predominantly unfavorable by the Shimada classification. In addition, for 17 patients tumor tissue was assessed for telomerase activity post-chemotherapy. In those cases where the tumor was negative for telomerase activity before and after chemotherapy, the patients uniformly did well. In cases where the tumor was positive before and negative or weakly positive after treatment, two of the seven patients did well clinically. However, in cases that were positive after chemotherapy, all had recurrence or died. In conclusion, telomerase activity appears to be a prognostic factor for neuroblastomas. In addition, assessment of tumors post-chemotherapy may be a further indicator of clinical outcome. PMID: 11200492 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 111: Cancer Lett. 2001 Jan 26;162(2):237-43. DAP-5 is involved in MycN/IFNgamma-induced apoptosis in human neuroblastoma cells. Wittke I, Madge B, Wiedemeyer R, Kimchi A, Schwab M. Department of Cytogenetics-H0400, German Cancer Research Center, Im Neuenheimer Feld 280, D 69120, Heidelberg, Germany. Death associated protein-5 (DAP-5) is a ubiquitously expressed member of the translation initiation factor eIF4G family that lacks the eIF4E binding site. A dominant negative fragment of DAP-5 protects HeLa cells from IFNgamma-induced cell death. By employing a functional approach we examined the role of DAP-5 in human neuroblastoma cells that are sensitized for IFNgamma-induced apoptosis by tetracycline controlled MYCN expression. DAP-5 fragment transcribed at high levels and translated into a functional miniprotein of 28 kDa protected neuroblastoma cells from IFNgamma-induced apoptosis. Reduced serum levels were toxic to cells constitutively expressing DAP-5 fragment suggesting that DAP-5 protein is essential for both viability and death of human neuroblastoma cells. PMID: 11146231 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 112: Genes Chromosomes Cancer. 2001 Feb;30(2):168-74. Chromosome bands 1p35-36 contain two distinct neuroblastoma tumor suppressor loci, one of which is imprinted. Caron H, Spieker N, Godfried M, Veenstra M, van Sluis P, de Kraker J, Voute P, Versteeg R. Institute of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. H.N.Caron@AMC.UVA.NL A previous loss of heterozygosity (LOH) study of a series of 91 neuroblastomas suggested that the 1p35-36 region encodes at least two tumor suppressor genes (TSGs) of importance in neuroblastoma development. Here we present the results of a study including 205 neuroblastomas that were analyzed for LOH at chromosome 1 and MYCN amplification. The results corroborate the existence of two TSGs on 1p. Distinct 1p loci seem to be involved in MYCN single copy vs. MYCN amplified neuroblastoma, as these tumors display a different type of shortest region of overlap (SRO). About 15% of MYCN single copy neuroblastomas show 1p deletions of variable length with an SRO of 47 cR at 1p36.3. The lost alleles are preferentially of maternal origin (P = 0.0002), suggesting parental imprinting of the locus. MYCN amplified neuroblastomas have a contrasting pattern of 1p loss. These tumors display much larger deletions of at least 89 cR comprising the region from 1p36.1 to the telomere. LOH of 1p is detected in 86% of the cases. The lost alleles are of random parental origin, suggesting inactivation of a non-imprinted TSG. Copyright 2000 Wiley-Liss, Inc. PMID: 11135433 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 113: Genes Chromosomes Cancer. 2001 Jan;30(1):87-90. MYCN amplification and 17q in neuroblastoma: evidence for structural association. O'Neill S, Ekstrom L, Lastowska M, Roberts P, Brodeur GM, Kees UR, Schwab M, Bown N. Cytogenetics Unit, School of Biochemistry and Genetics, University of Newcastle Upon Tyne, United Kingdom. MYCN oncogene amplification in neuroblastoma is statistically associated with gain of chromosome segment 17q21-qter. In neuroblastoma cell lines and primary tumors with MYCN amplification in the form of homogeneously staining regions (hsrs), juxtaposition of chromosome 17 material with MYCN sequences has occasionally been reported, raising the possibility of a physical affinity between MYCN and chromosome arm 17q. We used FISH to test for association between chromosome 17 segments and MYCN in eight neuroblastoma cell lines and two neuroblastoma primary tumors known to include hsrs. Evidence of an association was found in the chromosomes of both primary tumors; in one, a MYCN hsr was inserted into a structurally abnormal chromosome 17, in the other, an hsr in 16p was shown to be flanked by 17 material. In cell line NCG, hsrs in 4q and 16p were flanked by 17q material. These observations confirm the juxtaposition of 17q material with MYCN sequences in some neuroblastomas, and imply that there may be a physical or functional relationship between these two features in MYCN amplified neuroblastoma. Publication Types: Case Reports PMID: 11107180 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 114: Med Pediatr Oncol. 2000 Dec;35(6):669-72. Differential expression in an antisense MYCN neuroblastoma model. Schmidt ML, Lal A, Dittmann D. Department of Pediatrics, University of Illinois at Chicago, Chicago, Illinois, USA. mls3@uic.edu BACKGROUND: A previously published antisense MYCN-expressing model system was utilized to identify genes whose expression is altered by the down-regulation of MYCN by AS MYCN. RESULTS: Differential display comparing nontransfected human NB NBL-S cells to three AS MYCN-expressing cell lines (NBAS-4, -5, and -6) yielded nine differentially expressed cDNAs designated NDDE:1-9, for MYCN-dependent differential expression genes. A GenBank search revealed matches for seven of the nine cDNAs. Differential expression was confirmed for five of the cDNAs by Northern blot analysis. RESULTS: NDDE:8 is up-regulated in the AS MYCN-expressing clones and shares homology with the EB1 clone p53-induced gene (PRG3). NDDE:2 is up-regulated in the high-expressing N-myc protein NBL-S cells and shares homology with a 27 kD heat shock protein PAN1 clone. Further analysis of all five cDNAs might further elucidate the targets of MYCN in NB. Copyright 2000 Wiley-Liss, Inc. PMID: 11107143 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 115: Med Pediatr Oncol. 2000 Dec;35(6):656-8. Prognostic significance of EPHB6, EFNB2, and EFNB3 expressions in neuroblastoma. Tang XX, Zhao H, Robinson ME, Cnaan A, London W, Cohn SL, Cheung NK, Brodeur GM, Evans AE, Ikegaki N. Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318, USA. BACKGROUND: EPH family receptor tyrosine kinases and their ligand ephrins play pivotal roles in development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6), its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is predictive of favorable disease outcome of neuroblastoma (NB). When combined with TrkA expression, the expression of EPHB6, EFNB2, or EFNB3 predicts more accurately the disease outcome than each of the four variables alone. PROCEDURE: Cox regression and Kaplan-Meier analyses were used to assess the prognostic significance of EPHB6, EFNB2, EFNB3, and TrkA expressions in NB without MYCN amplification. RESULTS: High-level expression of EFNB3 or TrkA predicted favorable NB outcome of NB without MYCN amplification (p < 0.03). As found in the general NB population, EPHB6, EFNB2, or EFNB3 expression in combination with TrkA expression was significantly predictive of the disease outcome of normal MYCN NB (p < 0.01). CONCLUSIONS: EPHB6, EFNB2, and EFNB3 expressions may permit further refinement of the prognostic stratification of NB into favorable and unfavorable groups. Copyright 2000 Wiley-Liss, Inc. PMID: 11107140 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 116: Med Pediatr Oncol. 2000 Dec;35(6):623-7. Different effects of TrkA expression in neuroblastoma cell lines with or without MYCN amplification. Eggert A, Ho R, Ikegaki N, Liu XG, Brodeur GM. Division of Oncology, The Children's Hospital of Philadelphia and Department of Pediatrics, The University of Pennsylvania, Philadelphia, Pennsylvania 19104-4318, USA. BACKGROUND: Expression of the neurotrophin receptor TrkA is associated with a favorable prognosis in neuroblastoma (NB) and promotes growth inhibition and neuronal differentiation. Aggressive, MYCN-amplified NB tumors express little or no TrkA mRNA, suggesting that MYCN overexpression may inhibit TrkA expression. PROCEDURE: To study the interactions of TrkA expression and MYCN amplification in NB, we stably expressed the TrkA receptor in the MYCN single copy cell lines SH-SY5Y and NB69 as well as in the MYCN amplified cell lines CHP134 and IMR5. RESULTS: All four transfected cell lines demonstrated high TrkA expression and similar activation of the TrkA receptor and of mitogen-activated protein kinases as well as induction of immediate-early genes in response to nerve growth factor (NGF). Introduction of TrkA restored NGF responsiveness of SH-SY5Y and NB69 cells, as demonstrated by morphologic differentiation, growth inhibition, and enhanced survival in serum-free medium. However, no morphologic, growth, or survival responses to NGF were detected in MYCN-amplified CHP134 and IMR5 TrkA transfectants. CONCLUSIONS: Thus, transfection of TrkA into MYCN amplified NB cell lines only partly restored the TrkA/NGF signaling pathway, suggesting additional inhibitory effects of MYCN overexpression on TrkA signaling. Copyright 2000 Wiley-Liss, Inc. PMID: 11107132 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 117: Med Pediatr Oncol. 2000 Dec;35(6):597-602. Retinoic-acid-resistant neuroblastoma cell lines show altered MYC regulation and high sensitivity to fenretinide. Reynolds CP, Wang Y, Melton LJ, Einhorn PA, Slamon DJ, Maurer BJ. Division of Hematology-Oncology, Childrens Hospital Los Angeles, Los Angeles, California 90027, USA. preynolds@chla.usc.edu BACKGROUND: High-dose, pulse-13-cis-retinoic acid (13-cis-RA) given after intensive cytotoxic therapy improves event-free survival for high-risk neuroblastoma (NB), but more than 50% of patients have tumor recurrence. PROCEDURE: We conducted multistep selection for resistance to all-trans-retinoic acid (ATRA) in NB cell lines with (SMS-KCNR and LA-N-5) or without (SMS-LHN) MYCN genomic amplification. RESULTS: After 12 exposures to 10 microM ATRA, the two MYCN-amplified cell lines (KCNR 12X RR and LA-N-5 12X RR) showed partial resistance to the cytostatic/differentiation effects of ATRA; complete resistance was seen in LHN 12X RR. ATRA-selected cells showed general RA resistance (cross-resistance to 13-cis-RA). Transient (KCNR 12 X RR, LA-N-5 12X RR) or sustained (LHN 12X RR) novel overexpression of c-myc was associated with RA resistance. RA-insensitive overexpression of MYCN by transduction in SMS-LHN also conferred RA resistance. Both parental and RA-resistant lines showed 2-4 logs of cell kill in response to N-(4-hydroxyphenyl)retinamide (4- HPR, fenretinide). Compared to parental lines, 4-HPR achieved 1-3 log greater cell kills in RA-resistant LHN 12X RR, LA-N-5 12X RR, KCNR 12X RR, and MYCN-transduced SMS-LHN or SK-N-RA. NB cell lines (n = 26) from 21 different patients showed that 16 of 26 (62%) were sensitive to 4-HPR (LC(90) < 10 microM), including lines established at relapse after myeloablative and/or 13-cis-RA therapy. CONCLUSION: Thus, RA-resistant NB cell lines can be sensitive (and in some cases collaterally hypersensitive) to 4-HPR. Copyright 2000 Wiley-Liss, Inc. PMID: 11107126 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 118: Med Pediatr Oncol. 2000 Dec;35(6):582-4. MycN sensitizes neuroblastoma cells for drug-triggered apoptosis. Fulda S, Lutz W, Schwab M, Debatin KM. University Children's Hospital, Ulm, Germany. BACKGROUND: Amplification of the MYCN gene is found in a large proportion of neuroblastomas and is associated with a poor prognosis. PROCEDURE: To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs, we used a human neuroblastoma cell line with tetracycline-controlled expression of MycN. RESULTS: Neither conditional expression of MycN alone nor low drug concentrations induced apoptosis. However, MycN and cytotoxic drugs cooperated to induce cell death. Apoptosis triggered by MycN and doxorubicin was mediated by cleavage of caspases and involved activation of the CD95 system. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression and to trigger mitochondrial permeability transition and cytochrome c release. CONCLUSION: In that amplification of MYCN is considered an adverse prognostic factor, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited. Copyright 2000 Wiley-Liss, Inc. PMID: 11107122 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 119: Med Pediatr Oncol. 2000 Dec;35(6):559-62. BIN1 inhibits colony formation and induces apoptosis in neuroblastoma cell lines with MYCN amplification. Hogarty MD, Liu X, Thompson PM, White PS, Sulman EP, Maris JM, Brodeur GM. Department of Pediatrics, University of Pennsylvania School of Medicine and The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318, USA. hogartym@email.chop.edu BACKGROUND: MYCN amplification and overexpression occurs in 25% of neuroblastomas and independently predicts for poor prognosis disease, an effect thought to be mediated by its role as a transcriptional activator of growth promoting genes. However, in many mammalian cells, deregulated expression of MYC family genes (including MYCN) induces apoptosis. We hypothesized that BIN1, a MYC interacting protein capable of inducing apoptosis, may be an important regulator of MYCN in neuroblastoma. RESULTS: BIN1 expression was found to be reduced in MYCN-amplified cell lines. Further, forced expression of BIN1 markedly reduced colony formation in MYCN-amplified, but not single-copy, cell lines. This effect appeared to be caused by an increase in apoptosis, and was augmented by serum deprivation and concurrent cytotoxic drug therapy in cell culture CONCLUSION: BIN1 inactivation may be necessary for MYCN overexpression to lead to cellular proliferation rather than programmed cell death in neuroblastomas with MYCN amplification. Copyright 2000 Wiley-Liss, Inc. PMID: 11107117 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 120: Med Pediatr Oncol. 2000 Dec;35(6):544-6. Deletion of 11q23 is a frequent event in the evolution of MYCN single-copy high-risk neuroblastomas. Guo C, White PS, Hogarty MD, Brodeur GM, Gerbing R, Stram DO, Maris JM. Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA. BACKGROUND: Deletions of the long arm of chromosome 11 are frequently identified in human neuroblastomas. PROCEDURE: We screened 394 primary neuroblastomas and 52 tumor-derived cell lines with a panel of 11q and 11p polymorphic markers to determine the frequency of chromosome 11 allelic deletion, and to differentiate partial deletions of chromosome 11q (unb[11q] LOH) from whole chromosome loss. RESULTS: Allelic deletion occurred most frequently at cytogenetic band 11q23 and was detected in 161 primary neuroblastomas (41%) and 18 cell lines (35%). Eighty-seven tumors (22%) had unb[11q] LOH with a heterogeneous distribution of deletion breakpoints. Unb[11q] LOH was highly correlated with age > 1 year at diagnosis (P = 0.008), stage 4 disease (P = 0.001), unfavorable Shimada histopathology (P < 0.001), and assignment to a high-risk therapeutic protocol (P < 0.001), and was inversely correlated with MYCN amplification (P = 0.018). Patients whose tumors showed unb[11q] LOH were less likely to survive (P < 0.001), but there was only a trend towards an independent prognostic influence in multivariate analyses. CONCLUSIONS: Thus, structural rearrangements resulting in unb[11q] LOH commonly occur during the malignant evolution of high-risk neuroblastomas with single-copy MYCN. Copyright 2000 Wiley-Liss, Inc. PMID: 11107113 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 121: Genes Chromosomes Cancer. 2000 Dec;29(4):297-308. Topology of double minutes (dmins) and homogeneously staining regions (HSRs) in nuclei of human neuroblastoma cell lines. Solovei I, Kienle D, Little G, Eils R, Savelyeva L, Schwab M, Jager W, Cremer C, Cremer T. Institute for Anthropology and Humangenetics, University of Munich (LMU), Munich, Germany. Amplification of the MYCN gene is a characteristic feature of many neuroblastomas and is correlated with aggressive tumor growth. Amplicons containing this gene form either double minutes (dmins) or homogeneously staining regions (HSRs). To study the nuclear topology of these tumor-specific and transcriptionally active chromatin structures in comparison to chromosome territories, we performed fluorescence in situ hybridization with a MYCN probe and various chromosome paint probes, confocal laser scanning microscopy, and quantitative three-dimensional image analysis. The dmins formed dot-like structures in interphase nuclei and were typically located at the periphery of complexly folded chromosome territories; dmins noted in the chromosome territory interior were often detected within an invagination of the territory surface. Interphase HSRs typically formed extremely expanded structures, which we have never observed for chromosome territories of normal and tumor cell nuclei. Stretches of HSR-chromatin often extended throughout a large part of the cell nucleus, but appeared well separated from neighboring chromosome territories. We hypothesize that dmins are located within the interchromosomal domain (ICD) space and that stretches of HSR-chromatin align along this space. Such a topology could facilitate access of amplified genes to transcription and splicing complexes that are assumed to localize in the ICD space. Copyright 2000 Wiley-Liss, Inc. PMID: 11066073 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 122: Cancer Genet Cytogenet. 2000 Sep;121(2):139-45. Concomitant amplification and expression of PAX7-FKHR and MYCN in a human rhabdomyosarcoma cell line carrying a cryptic t(1;13)(p36;q14). Frascella E, Lenzini E, Schafer BW, Brecevic L, Dorigo E, Toffolatti L, Nanni P, De Giovanni C, Rosolen A. Department of Pediatrics, University of Padua, Padua, Italy. Alveolar rhabdomyosarcoma (ARMS) is associated with the specific chromosomal translocation (2;13)(q35;q14) or its rarer variant t(1;13)(p36;q14), which produces the fusion gene PAX7-FKHR. Here we describe the human cell line RC2, derived from an ARMS, which harbors a cryptic t(1;13)(p36;q14) and concomitantly shows amplification of the PAX7-FKHR fusion gene and of the MYCN oncogene. The t(1;13) and MYCN oncogene were studied by standard cytogenetic analysis and molecular techniques. The reverse transcriptase polymerase chain reaction demonstrated the expression of PAX7-FKHR mRNA in RC2 cells, although karyotype analysis failed to demonstrate a t(1;13)(p36;q14) chromosomal translocation or a derivative 13 chromosome. Double minute chromosomes were detected in all the metaphases studied. Fluorescence in situ hybridization analysis revealed multiple copies of the PAX7-FKHR fusion gene localized exclusively on a subset of double minutes, whereas multiple copies of MYCN were identified on other double minute chromosomes. Southern-blot analysis demonstrated that RC2 cells contain approximately 20 copies of the MYCN oncogene. So far no continuous RMS cell line carrying the t(1;13)(p36;q14) has been described, and PAX7-FKHR and MYCN amplifications have always been reported to occur separately in rhabdomyosarcoma (RMS). The availability of an ARMS cell line that harbors the t(1;13)(p36;q14) constitutes a useful tool for further understanding the role of the PAX7-FKHR fusion gene in RMS oncogenesis and may improve knowledge of the possible relation between PAX7-FKHR and MYCN amplification. PMID: 11063797 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 123: J Clin Oncol. 2000 Nov 1;18(21):3604-13. Comment in: J Clin Oncol. 2000 Nov 1;18(21):3591-4. MYCN expression is not prognostic of adverse outcome in advanced-stage neuroblastoma with nonamplified MYCN. Cohn SL, London WB, Huang D, Katzenstein HM, Salwen HR, Reinhart T, Madafiglio J, Marshall GM, Norris MD, Haber M. Department of Pediatrics, Northwestern University Medical School, Chicago, Illinois, USA. scohn@northwestern.edu PURPOSE: The clinical significance of MYCN expression in children with neuroblastoma (NB) remains controversial. To determine the prognostic significance of MYCN expression in the absence of MYCN amplification, we analyzed MYCN mRNA and protein expression in tumors from 69 patients. PATIENTS AND METHODS: Sixty-nine NB tumor samples with nonamplified MYCN from patients with stage C or D disease were obtained from the Pediatric Oncology Group Neuroblastoma Tumor Bank. MYCN mRNA was analyzed using a real-time reverse transcriptase polymerase chain reaction assay, and MYCN protein was examined by Western blot analyses. RESULTS: The estimated 5-year event-free survival (EFS) and survival (S) rates plus SE for the cohort were 57% +/- 17% and 60% +/- 16%, respectively. Infants younger than 1 year had significantly higher rates of EFS and S than children >/= 1 year of age (P =.003 and P <.001, respectively); patients with stage C disease had better outcome than those with stage D NB (P <.001); and patients with hyperdiploid tumors had better outcome than those with diploid NB (P <.001). Surprisingly, outcome was slightly better for patients with high versus low levels of MYCN mRNA expression (4-year S, 70% +/- 13% v 50% +/- 16%; P =.290), and for patients with tumors that expressed MYCN protein (4-year S, 73% +/- 19% v 53% +/- 15%, respectively; P =.171). CONCLUSION: High levels of MYCN expression are not prognostic of adverse outcome in patients with advanced-stage NB with nonamplified MYCN. A trend associating high levels of MYCN expression with improved outcome was observed. PMID: 11054433 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 124: J Clin Oncol. 2000 Nov 1;18(21):3591-4. Comment on: J Clin Oncol. 2000 Nov 1;18(21):3604-13. MYCN expression in neuroblastoma: A mixed message? Matthay KK. Publication Types: Comment Editorial PMID: 11054431 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 125: Exp Cell Res. 2000 Nov 1;260(2):396-403. MYCN-related suppression of functional CD44 expression enhances tumorigenic properties of human neuroblastoma cells. Gross N, Balmas Bourloud K, Brognara CB. Onco-Hematology Unit, University Hospital CHUV, Lausanne, 1011, Switzerland. Nicole.Gross@chuv.hospvd.ch Highly malignant neuroblastoma tumors with MYCN amplification have been shown to downregulate the expression of the CD44 adhesion receptor. We have previously shown that MYCN amplified neuroblastoma cell lines either lack CD44 expression or express a nonfunctional, nonhyaluronic acid-binding CD44 receptor. By analysis of cells with manipulated expression of either CD44 or MYCN, we demonstrate that transfection of cells with a CD44 full-length cDNA construct produced a functional receptor in single copy MYCN cells and a nonfunctional CD44 receptor in MYCN amplified cells, similar to the CD44 receptor expressed by cells with enforced MYCN. Analysis of the in vivo growth properties of the transfectants revealed that the restoration of a functional CD44 receptor in nonamplified cells resulted in the suppression of in vivo cell growth, therefore linking the MYCN-related lack of hyaluronic acid-binding function of CD44 to the highly tumorigenic properties of a subset of neuroblastoma cells. Copyright 2000 Academic Press. PMID: 11035936 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 126: Neurosci Lett. 2000 Oct 13;292(3):147-50. Synergy between 5' and 3' flanking regions of the human tyrosine hydroxylase gene ensures specific, high-level expression in neuroblastoma cells. Gardaneh M, Gilbert J, Haber M, Norris MD, Cohn SL, Schmidt ML, Marshall GM. Children's Cancer Institute Australia for Medical Research, NSW, Randwick, Australia. Factors regulating tyrosine hydroxylase (TH) gene transcription are of major importance in the studies of malignant and degenerative diseases of catecholamine-synthesizing tissues. In this study, we used transient transfection of a reporter gene to show that high-level, tissue-specific TH expression was only achieved when the reporter gene was cloned between a 5' TH promoter sequence (-513-+1), and, a 3' TH gene flanking sequence (end of exon 14-+976). We also show that TH mRNA expression level is closely linked to the expression level of the proto-oncogene, MYCN in neuroblastoma tumor cell lines. Taken together our data indicate that MYCN may regulate TH expression in neuroblastoma cells, but not through binding to the 5' or 3' TH gene flanking sequences used in our experiments. PMID: 11018298 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 127: Int J Cancer. 2000 Sep 20;89(5):418-22. Expression of glypican 3 (GPC3) in embryonal tumors. Saikali Z, Sinnett D. Division of Hematology-Oncology, Charles-Bruneau Cancer Center, Research Center, Sainte-Justine Hospital, Montreal, Quebec, Canada. Embryonal tumors, such as neuroblastoma, medulloblastoma and Wilms' tumor, have their peak incidence in the first 4 years of life. These neoplasias exhibit genetic and clinical heterogeneity, but little is known about their molecular pathogenesis. Application of the differential-display PCR approach led to the identification of a gene, glypican 3 (GPC3), that is differentially expressed in cancer cells. Expression of this gene is usually limited to fetal mesodermal tissue, and its inactivation has been found to be responsible for the X-linked Simpson-Golabi-Behmel overgrowth syndrome. Here, we show that GPC3 mRNA is present in several neuroblastomas and all Wilms' tumors tested to date but not in medulloblastoma. GPC3 was not expressed in normal kidney tissues obtained from the corresponding Wilms' tumor patients, suggesting that in these cancer cells expression was not repressed (or was activated). No correlation was found between expression of GPC3 and the known indicator of neuroblastoma prognosis MYCN mRNA. However, all samples that expressed GPC3 also expressed IGF-II, coding for a growth factor important in the survival and growth of many cancer types. Although the biological significance of this relationship remains unclear, our results suggest that GPC3 may be implicated in the development of embryonal tumors through a signaling pathway that appears to involve IGF-II. Copyright 2000 Wiley-Liss, Inc. PMID: 11008203 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 128: Br J Cancer. 2000 Oct;83(8):973-9. Adverse outcome of infants with metastatic neuroblastoma, MYCN amplification and/or bone lesions: results of the French society of pediatric oncology. Minard V, Hartmann O, Peyroulet MC, Michon J, Coze C, Defachelle AS, Lejars O, Perel Y, Bergeron C, Boutard P, Leverger G, Stephan JL, Thyss A, Chastagner P, Couillault G, Devalck C, Lutz P, Mechinaud F, Millot F, Plantaz D, Rialland X, Rubie H. Department of Pediatrics, Institut Gustave Roussy, 39 rue Camille Desmoulins, Villejuif Cedex, 94805, France. To assess the relevance of MYCN amplification and bone lesions in stage 4 neuroblastoma (NB) in infants aged <1 year, 51 infants with stage 4 NB were enrolled. Three groups of patients were defined according to the type of metastases and the resectability of the primary tumour. Group I comprised 21 infants with radiologically detectable bone lesions, Group II 22 patients with an unresectable primary tumour and Group III eight patients with only metaiodobenzylguanidine (MIBG) skeletal uptake. MYCN oncogene content was assayed in 47/51 tumours and found to be amplified in 17 (37%). The 5-year event-free survival (EFS) rate of these 51 infants was 64.1% (+/- 7.1%). In a univariate analysis, bone lesions, MYCN amplification, urinary vanillylmandelic/homovanillic acid ratio and serum ferritin levels adversely influenced outcome. In the multivariate analysis, radiologically detectable bone lesions were the most powerful unfavourable prognostic indicator: the EFS rate was 27.2% for these infants compared to 90% for infants without bone lesions (P<0.0001). Our data emphasize the poor prognosis of infants affected by stage 4 NB with bone lesions, especially when associated with MYCN amplification. Given the poor results in this group whatever the treatment, new therapeutic approaches need to be investigated in the future. Publication Types: Clinical Trial Controlled Clinical Trial Multicenter Study PMID: 10993641 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 129: Adv Exp Med Biol. 2000;476:239-48. MYCN oncogene and angiogenesis: down-regulation of endothelial growth inhibitors in human neuroblastoma cells. Purification, structural, and functional characterization. Hatzi E, Breit S, Zoephel A, Ashman K, Tontsch U, Ahorn H, Murphy C, Schweigerer L, Fotsis T. Lab. of Biological Chemistry, Medical School, University of Ioannina, Greece. Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates three inhibitors of endothelial cell proliferation. One of them was identified by amino acid sequencing as being identical with activin A, a developmentally-regulated protein. Down-regulation involves interaction of the N-myc protein with the activin A promoter. Work is ongoing to characterize the other two endothelial cell inhibitors. We suggest that the N-myc induced down-regulation of angiogenesis inhibitors could contribute to tumor angiogenesis. PMID: 10949669 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 130: Genes Chromosomes Cancer. 2000 Sep;29(1):1-8. CGH analysis of radon-induced rat lung tumors indicates similarities with human lung cancers. Dano L, Guilly MN, Muleris M, Morlier JP, Altmeyer S, Vielh P, El-Naggar AK, Monchaux G, Dutrillaux B, Chevillard S. CEA, DSV, DRR, Fontenay-aux-Roses, France. Epidemiological studies have shown that inhalation of radon, a radioactive gas, is associated with an increased risk for lung cancer. We have developed a model of radon-induced rat lung tumors to characterize cytogenetic and molecular events involved in radon-induced lung tumorigenesis. Using comparative genomic hybridization (CGH), gains and losses of genetic material were investigated in a series of 13 carcinomas and four adenomas of the lung. Frequent losses occurred at 4q12-21, 5q11-33, and 15q, which are homologous to human chromosome (HSA) bands 7q21-36, 1p31-36/9p21-31, and 13q14.1-14.3/3p14.2, respectively. These regions are frequently (30-80%) deleted in human lung cancer and contain tumor suppressor genes or proto-oncogenes such as MET, CDKN2A/p16/MTS1, CDKN2B/p15/MTS2, FHIT, and RB1 or yet to be identified genes. Frequent gains involved 6, 7q34-qter, and 19q; chromosomes 6 and 7 being homologous to human 2p21-25 and 8q21-24 where the MYCN and MYC oncogenes are located. The genetic similarities between rat and human lung cancer suggest common underlying mechanisms for tumor evolution in both species. Moreover, cytogenetic and molecular genetic analyses of radon-induced rat lung tumors could help to better understand the development and progression of radon-induced lung cancer in man. Copyright 2000 Wiley-Liss, Inc. PMID: 10918387 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 131: J Clin Oncol. 2000 Jul;18(13):2582-92. Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: a molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues. Poremba C, Scheel C, Hero B, Christiansen H, Schaefer KL, Nakayama J, Berthold F, Juergens H, Boecker W, Dockhorn-Dworniczak B. Gerhard-Domagk-Institute of Pathology and Department of Pediatric Hematology and Oncology, Westfalische Wilhelms-University, Munster, Germany. PURPOSE: We have recently demonstrated that telomerase activity (TA) is an independent prognostic factor in neuroblastomas. In the present study, the prognostic impact of TA and gene expression of the three major telomerase subunits is evaluated by molecular and immunohistochemical techniques in fresh-frozen and paraffin-embedded tissues. PATIENTS AND METHODS: One hundred thirty-three neuroblastomas of all stages were analyzed for TA. The TA levels of 75 neuroblastoma cases were correlated with gene expression of telomerase subunits hTRT, human telomerase RNA (hTR), and telomerase protein 1 (TP1) by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), using an innovative approach on the LightCycler instrument (Roche Diagnostics, Mannheim, Germany). For selected cases, the applicability of RT-PCR and immunohistochemistry for hTRT expression analysis was investigated in paraffin-embedded tissues. TA and subunit expression patterns were correlated with traditional prognostic indicators and disease outcome. RESULTS: TA was present in a total of 39 (29.3%) of 133 neuroblastomas and in 31 (29.8%) of 104 initial neuroblastomas without cytotoxic pretreatment. TA was significantly correlated with both event-free and overall survival (P <.0001). Furthermore, we found a significant correlation between expression levels of TA and hTRT (P <.0001) as well as hTR (P <.001). Multivariate analysis revealed only TA and tumor stage but not serum lactate dehydrogenase, MYCN amplification, or age at diagnosis as independent prognostic factors. CONCLUSION: The significant correlation with clinical outcome strongly recommends that analysis of TA be incorporated into the clinical investigation of each individual neuroblastoma at the time of diagnosis. Because the mere presence or absence of TA without further quantification is sufficient basis for predicting disease outcome, the telomeric repeat amplification protocol assay could be complemented with but not replaced by analysis of hTRT or hTR expression. PMID: 10893290 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 132: Br J Cancer. 2000 Jul;83(1):40-9. Fluorescence in situ hybridization techniques for the rapid detection of genetic prognostic factors in neuroblastoma. United Kingdom Children's Cancer Study Group. Taylor CP, Bown NP, McGuckin AG, Lunec J, Malcolm AJ, Pearson AD, Sheer D. Human Cytogenetics Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK. Neuroblastoma is the commonest extracranial solid tumour in children. There are a number of molecular genetic features known which are of prognostic importance and which are used to direct therapy. Identification and targeting of high-risk individuals with intensive therapeutic regimens may allow an improvement in survival rates. The most powerful biological parameters associated with prognosis in this malignancy are chromosomal changes, especially MYCN amplification, deletion of chromosome 1p and aneuploidy. Rapid characterization of these aberrations at the time of diagnosis is paramount if stratification according to risk group is to be achieved. This paper describes the rapid detection of del(1p), MYCN amplification and trisomy using interphase fluorescence in situ hybridization on imprints from fresh tumour biopsies. The results are related to those obtained by standard molecular methods and karyotyping. PMID: 10883666 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 133: J Med Genet. 2000 Jul;37(7):501-9. Gene amplification in PNETs/medulloblastomas: mapping of a novel amplified gene within the MYCN amplicon. Fruhwald MC, O'Dorisio MS, Rush LJ, Reiter JL, Smiraglia DJ, Wenger G, Costello JF, White PS, Krahe R, Brodeur GM, Plass C. Division of Pediatric Hematology and Oncology, The Ohio State University and the Comprehensive Cancer Center, Columbus 43210, USA. fruhwald@uni-muenster.de OBJECTIVES: The pathological entity of primitive neuroectodermal tumour/medulloblastoma (PNET/MB) comprises a very heterogeneous group of neoplasms on a clinical as well as on a molecular level. We evaluated the importance of DNA amplification in medulloblastomas and other primitive neuroectodermal tumours (PNETs) of the CNS. METHOD: Restriction landmark genomic scanning (RLGS), a method that allows the detection of low level amplification, was used. RLGS provides direct access to DNA sequences circumventing positional cloning efforts. Furthermore, we analysed several samples by CGH. DESIGN: Twenty primary medulloblastomas, five supratentorial PNETs, and five medulloblastoma cell lines were studied. RESULTS: Although our analysis confirms that gene amplification is generally a rare event in childhood PNET/MB, we found a total of 17 DNA fragments that were amplified in seven different tumours. Cloning and sequencing of several of these fragments confirmed the previous finding of MYC amplification in the cell line D341 Med and identified novel DNA sequences amplified in PNET/MB. We describe for the first time amplification of the novel gene, NAG, in a subset of PNET/MB. Despite genomic amplification, NAG was not overexpressed in the tumours studied. We have determined that NAG maps less than 50 kb 5' of DDX1 and approximately 400 kb telomeric of MYCN on chromosome 2p24. CONCLUSION: We found a similar but slightly higher frequency of amplification than previously reported. We present several DNA fragments that may belong to the CpG islands of novel genes amplified in a small subset of PNET/MB. As an example we describe for the first time the amplification of NAG in the MYCN amplicon in PNET/MB. PMID: 10882752 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 134: J Pediatr Hematol Oncol. 2000 May-Jun;22(3):197-205. Natural history and biology of stage A neuroblastoma: a Pediatric Oncology Group Study. Alvarado CS, London WB, Look AT, Brodeur GM, Altmiller DH, Thorner PS, Joshi VV, Rowe ST, Nash MB, Smith EI, Castleberry RP, Cohn SL. Department of Pediatrics, Emory University, Atlanta, Georgia, USA. PURPOSE: To prospectively analyze the outcome of patients with Stage A neuroblastoma (NB) treated with surgery alone, especially with regard to the prognostic significance of age, tumor site, MYCN copy number, tumor cell ploidy, and histology. PATIENTS AND METHODS: The clinical course of 329 patients with Stage A disease registered on the POG NB Biology Study #9047 between February, 1990 and October, 1997 were evaluated. Age, tumor site, MYCN copy number, tumor cell ploidy, and histology were analyzed for their impact on event-free survival (EFS) and survival (S). RESULTS: The 5-year estimated EFS and S rates for the 329 patients were 91% (+/-3%) and 96% (+/-2%), respectively. The EFS rate was similar for infants younger than 12 months and children age 12 months or older, but age older than 12 months was predictive of lower S rates (P = 0.044). Patients with adrenal, abdominal non-adrenal, thoracic, and cervical tumors had similar S rates. The majority of patients had tumors with favorable biologic features, and only 3% had MYCN amplification. For infants with diploid tumors, the EFS rate was 82% (+/-16%), but effective therapy yielded an S rate of 100%. Rate of S was 80% (+/-26%) and 64% (+/-27%) for patients with unfavorable tumor histology and MYCN-amplified tumors, respectively. CONCLUSION: The outcome for patients with Stage A NB treated with surgery alone is excellent. Although EFS and S rates were significantly worse for patients with MYCN-amplified tumors, a subset achieved long-term remission after surgery alone. For patients with Stage A and MYCN amplification, additional factors are needed to distinguish the patients who will achieve long-term remission with surgery alone from those who will develop recurrent disease. PMID: 10864050 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 135: Genes Chromosomes Cancer. 2000 Jul;28(3):276-84. Molecular analysis of chromosome arm 17q gain in neuroblastoma. Janoueix-Lerosey I, Penther D, Thioux M, de Cremoux P, Derre J, Ambros P, Vielh P, Benard J, Aurias A, Delattre O. Laboratoire de Pathologie Moleculaire des Cancers (Unite INSERM 509), Institut Curie, Paris, France. Complete or partial gain of the long arm of chromosome 17 (17q) has been shown recently by molecular cytogenetic techniques to be the most frequent chromosomal change in neuroblastoma and to be associated with adverse prognosis. Few reports, however, have focused on the precise mapping of the commonly overrepresented region. We have investigated 17q gain by the analysis of allelic imbalances at microsatellite loci dispersed along chromosome 17 in a series of 69 neuroblastomas. Allelic imbalances for at least two consecutive loci were observed in 39/59 informative cases, that is in agreement with previously reported frequencies of 17q gain. In a subset of the cases, comparative genomic hybridization analysis established the relationship between these allelic imbalances and the gain of 17q material. A partial 17q gain was observed in 9 cases, delineating a common region of 17q gain between the marker D17S787 (75 cM, 360 cR) and the telomere. In most cases, molecular results were suggestive of partial tri- or tetrasomy, whereas in 4 cases a higher copy number was documented. Our results also confirm that the presence of additional 17q material is closely associated with 1p36 deletion, MYCN amplification, and diploid or tetraploid chromosomal content. Genes Chromosomes Cancer 28:276-284, 2000. Copyright 2000 Wiley-Liss, Inc. PMID: 10862033 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 136: Br J Cancer. 2000 Jun;82(11):1801-7. Detailed deletion mapping of chromosome band 14q32 in human neuroblastoma defines a 1.1-Mb region of common allelic loss. Hoshi M, Otagiri N, Shiwaku HO, Asakawa S, Shimizu N, Kaneko Y, Ohi R, Hayashi Y, Horii A. Department of Molecular Pathology, Tohoku University School of Medicine, Sendai, Japan. Neuroblastoma (NB) is a well-known malignant disease in infants, but its molecular mechanisms have not yet been fully elucidated. To investigate the genetic contribution of abnormalities on the long arm of chromosome 14 (14q) in NB, we analysed loss of heterozygosity (LOH) in 54 primary NB samples using 12 microsatellite markers on 14q32. Seventeen (31%) of 54 tumours showed LOH at one or more of the markers analysed, and the smallest common region of allelic loss was identified between D14S62 and D14S987. This region was estimated to be 1-cM long from the linkage map. Fluorescence in situ hybridization also confirmed the loss. There was no statistical correlation between LOH and any clinicopathologic features, including age, stage, amplification of MYCN and ploidy. We further constructed a contig spanning the lost region using bacterial artificial chromosome and estimated this region to be approximately 1.1-Mb by pulsed-field gel electrophoresis. Our results will contribute to cloning and characterizing the putative tumour-associated gene(s) in 14q32 in NB. PMID: 10839294 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 137: Cancer Res. 2000 May 1;60(9):2483-7. Genome-wide screen for allelic imbalance in a mouse model for neuroblastoma. Weiss WA, Godfrey T, Francisco C, Bishop JM. Department of Neurology, University of California, San Francisco 94143-0114, USA. weiss@cgl.ucsf.edu We have used the rat tyrosine hydroxylase promotor to overexpress MYCN in the neural crest of transgenic mice, resulting in a mouse model for neuroblastoma. Using PCR analysis of microsatellite markers, we conducted a genome-wide analysis in tumors from these animals. Regions of chromosomes 1, 3, 10, 11, 14, and 18 were affected in 20-50% of tumors. Analysis of a subset of these tumors by comparative genomic hybridization was consistent with the microsatellite data. The changes on mouse chromosomes 1, 11, 14, and 18 were syntenic with corresponding regions of loss of heterozygosity in human neuroblastoma, suggesting that genes implicated in the mouse tumors may also play a role in the pathogenesis of the human disease. One-third of the mouse tumors shared abnormalities on chromosomes 1, 3, and 10, whereas the remainder of tumors did not show this combination. These data suggest that genetic mutations on chromosomes 1, 3, and 10 cooperate in the pathogenesis of neuroblastoma and that neuroblastoma in the mouse arises from at least two distinct genetic pathways, one of which is dependent on lesions in chromosomes 1, 3, and 10, the other of which is not. PMID: 10811128 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 138: Nat Med. 2000 May;6(5):529-35. Comment in: Nat Med. 2000 May;6(5):498-500. Nat Med. 2002 Dec;8(12):1333-5; author reply 1335. Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Teitz T, Wei T, Valentine MB, Vanin EF, Grenet J, Valentine VA, Behm FG, Look AT, Lahti JM, Kidd VJ. Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38101, USA. Caspase 8 is a cysteine protease regulated in both a death-receptor-dependent and -independent manner during apoptosis. Here, we report that the gene for caspase 8 is frequently inactivated in neuroblastoma, a childhood tumor of the peripheral nervous system. The gene is silenced through DNA methylation as well as through gene deletion. Complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplification of the oncogene MYCN. Caspase 8-null neuroblastoma cells were resistant to death receptor- and doxorubicin-mediated apoptosis, deficits that were corrected by programmed expression of the enzyme. Thus, caspase 8 acts as a tumor suppressor in neuroblastomas with amplification of MYCN. PMID: 10802708 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 139: J Clin Oncol. 2000 May;18(9):1888-99. Loss of heterozygosity at 1p36 independently predicts for disease progression but not decreased overall survival probability in neuroblastoma patients: a Children's Cancer Group study. Maris JM, Weiss MJ, Guo C, Gerbing RB, Stram DO, White PS, Hogarty MD, Sulman EP, Thompson PM, Lukens JN, Matthay KK, Seeger RC, Brodeur GM. Department of Pediatrics, University of Pennsylvania School of Medicine and Children's Hospital of Philadelphia, Philadelphia, PA, USA. PURPOSE: To determine the independent prognostic significance of 1p36 loss of heterozygosity (LOH) in a representative group of neuroblastoma patients. PATIENTS AND METHODS: Diagnostic tumor specimens from 238 patients registered onto the most recent Children's Cancer Group phase III clinical trials were assayed for LOH with 13 microsatellite polymorphic markers spanning chromosome band 1p36. Allelic status at 1p36 was correlated with other prognostic variables and disease outcome. RESULTS: LOH at 1p36 was detected in 83 (35%) of 238 neuroblastomas. There was a correlation of 1p36 LOH with age at diagnosis greater than 1 year (P = .026), metastatic disease (P<.001), elevated serum ferritin level (P<.001), unfavorable histopathology (P<.001), and MYCN oncogene amplification (P<.001). LOH at 1p36 was associated with decreased event-free survival (EFS) and overall survival (OS) probabilities (P<.0001). For the 180 cases with single-copy MYCN, 1p36 LOH status was highly correlated with decreased EFS (P = .0002) but not OS (P = .1212). Entering 1p36 LOH into a multivariate regression model suggested a trend toward an independent association with decreased EFS (P = .0558) but not with decreased OS (P = .3687). Furthermore, allelic status at 1p36 was the only prognostic variable that was significantly associated with decreased EFS in low-risk neuroblastoma patients (P = .0148). CONCLUSION: LOH at 1p36 is independently associated with decreased EFS, but not OS, in neuroblastoma patients. Determination of 1p36 allelic status may be useful for predicting which neuroblastoma patients with otherwise favorable clinical and biologic features are more likely to have disease progression. Publication Types: Multicenter Study PMID: 10784629 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 140: Cancer Res. 2000 Apr 1;60(7):1908-13. Lysis of MYCN-amplified neuroblastoma cells by MYCN peptide-specific cytotoxic T lymphocytes. Sarkar AK, Nuchtern JG. Department of Surgery, Baylor College of Medicine, and Texas Children's Cancer Center, Texas Children's Hospital, Houston 77030-2399, USA. The effectiveness of cell-mediated immunotherapy for cancer can be limited by loss-of-antigen mutations that occur during tumor growth. In neuroblastoma, amplification of the MYCN oncogene correlates with rapid tumor progression and a poor prognosis overall. We propose that the MYCN protein, the high-level expression of which is required for maintenance of the malignant phenotype, would be an ideal target for vaccine therapy. The MYCN-derived S9K peptide (amino acids 7-15; STMPGMICK), which contains an HLA-A1 binding motif, was used to generate CTLs from the peripheral blood lymphocytes of an HLA-A1+ healthy donor and an HLA-A1+ patient with MYCN-amplified neuroblastoma These CTL lines specifically lysed HLA-matched, MYCN-amplified neuroblastoma tumor cells. They did not lyse either HLA-mismatched, MYCN-amplified, or matched/nonmatched, non-MYCN-amplified tumor cells. The CTL activity was inhibited by a monoclonal antibody to a class I HLA monomorphic determinant but not by one specific for HLA class II, consistent with a class I-restricted mechanism of cytotoxicity. Antibodies to CD8, but not those to CD4, also inhibited CTL activity, identifying CD8+ lymphocytes as the effector cell population. These results show that MYCN-derived peptides can serve as tumor-specific antigens and suggest a rational approach to cell-mediated immunotherapy for MYCN-amplified neuroblastoma. PMID: 10766179 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 141: Cancer. 2000 Apr 15;88(8):1955-63. Multifocal neuroblastoma: biologic behavior and surgical aspects. Hiyama E, Yokoyama T, Hiyama K, Yamaoka H, Matsuura Y, Nishimura S, Ueda K. Department of General Medicine, Hiroshima University, Faculty of Medicine, School of Medicine, Hiroshima, Japan. BACKGROUND: Although multifocal neuroblastoma is rare, its incidence has increased because of recent improvements in diagnostic tools and the introduction of mass screening. Among the 106 neuroblastoma cases treated at the authors' hospital between 1984 and 1998, 8 were multifocal neuroblastoma. METHODS: The authors examined clinicopathologic findings and biologic features, including MYCN amplification, NTRK1 and Ha-ras p21 expression, cellular DNA content, and telomerase activity in these 8 multifocal neuroblastoma cases. Moreover, clinicopathologic findings were investigated with a review of 53 published cases of multiple neuroblastoma in the literature published in English between 1966 and 1999. RESULTS: Among these eight cases, five were detected by mass screening and three were incidental neuroblastomas. Histologically, all tumors were classified as ganglioneuroma or favorable neuroblastoma except one advanced case. All tumors lacked the MYCN gene amplification and expressed NTRAK1 mRNA and Ha-ras p21 protein. Cellular DNA content showed that half of these tumors were near-triploid, and the proliferative index (%S-phase) of all tumors was less than 25%. High telomerase activity was detected in none of these cases. Four patients underwent multistage operation and five patients with bilateral adrenal neuroblastomas underwent tumor enucleation to preserve adrenal function. Currently, all patients are disease free and none have required corticosteroid replacement therapy. Among the previously reported 53 cases with multifocal neuroblastoma, 25 were incidentally detected, 18 had familiar history, and most patients without other major complications also had extremely good prognoses. CONCLUSIONS: These findings suggested that most multifocal neuroblastomas have favorable biologic features. Clinically, surgical approaches should be attempted to preserve organ function, especially adrenal function, and minimal invasive surgery should be performed. In cases of thoracoabdominal neuroblastoma, multistage surgery is effective and safe. Copyright 2000 American Cancer Society. Publication Types: Case Reports PMID: 10760774 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 142: J Clin Oncol. 2000 Mar;18(6):1260-8. Biologic factors determine prognosis in infants with stage IV neuroblastoma: A prospective Children's Cancer Group study. Schmidt ML, Lukens JN, Seeger RC, Brodeur GM, Shimada H, Gerbing RB, Stram DO, Perez C, Haase GM, Matthay KK. Department of Pediatrics, University of Illinois at Chicago College of Medicine, Chicago, IL, USA. mls3@uic.edu PURPOSE: A prospective Children's Cancer Group study, CCG-3881, has been completed to determine if a more accurate prediction of prognosis by biologic features can identify subgroups of infants with stage IV neuroblastoma (NBL) who require differing intensities of treatment. PATIENTS AND METHODS: One hundred thirty-four infants were registered from June 1989 to August 1995, with a median follow-up of 47.1 months (range, 0 to 88 months). The biologic factors examined were tumor MYCN copy number, Shimada histopathologic classification, serum ferritin, and bone marrow immunocytology (sensitivity, one tumor cell per 10(5) bone marrow cells). Patients treated on CCG-3881 (n = 116) received four-drug chemotherapy for 9 months (cisplatin, cyclophosphamide, doxorubicin, and etoposide), with surgery and local radiation to residual disease. After January 1991, all subsequent infants with tumor MYCN amplification (n = 18) were transferred after one cycle of therapy to the high-risk CCG-3891 protocol (open January 1991 to April 1996) for more intensive treatment. RESULTS: The 3-year event-free survival (EFS) and overall survival (mean +/- SD) for the 134 infants were 63% +/- 5% and 71% +/- 5%, respectively. Patients whose tumors were without MYCN amplification had a 93% +/- 4% 3-year EFS, whereas those with amplified MYCN had a 10% +/- 7% 3-year EFS (P <. 0001). Each of the other biologic features studied had prognostic significance in univariate analysis but not after stratifying by MYCN copy number. CONCLUSION: Infants less than 1 year of age at diagnosis with stage IV NBL have a much improved outcome compared with children >/= 1 year of age. Nonamplified MYCN tumors identify a group of infants with a 93% +/- 4% EFS, whereas amplified MYCN copy number clearly identifies patients who are unlikely to survive despite intensive chemotherapy. PMID: 10715296 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 143: Cancer Res. 2000 Feb 15;60(4):799-802. Detection of gene amplification by genomic hybridization to cDNA microarrays. Heiskanen MA, Bittner ML, Chen Y, Khan J, Adler KE, Trent JM, Meltzer PS. Cancer Genetics Branch, National Human Genome Research Institute, NIH, Bethesa, Maryland 20892, USA. Gene amplification is one of the major mechanisms of oncogene activation in tumorigenesis. To facilitate the identification of genes mapping to amplified regions, we have used a technique based on the hybridization of total genomic DNA to cDNA microarrays. To aid detection of the weak signals generated in this complex hybridization, we have used a tyramide-based technique that allows amplification of a fluorescent signal up to 1000-fold. Dilution experiment suggests that amplifications of 5-fold and higher can be detected by this approach. The technique was validated using cancer cell lines with several known gene amplifications, such as those affecting MYC, MYCN, ERBB2, and CDK4. In addition to the detection of the known amplifications, we identified a novel amplified gene, ZNF133, in the neuroblastoma cell line NGP. Hybridization of NGP cDNA on an identical array also revealed over expression of ZNF133. Parallel analysis of genomic DNA for copy number and cDNA for expression now provides rapid approach to the identification of amplified genes and chromosomal regions in tumor cells. PMID: 10706083 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 144: Lab Invest. 2000 Feb;80(2):271-3. Detection of MYCN amplification in neuroblastoma using competitive PCR quantitation. Oude Luttikhuis ME, Iyer VK, Dyer S, Ramani P, McConville CM. Division of Medical and Molecular Genetics, University of Birmingham, United Kingdom. PMID: 10701696 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 145: Pathology. 1999 Nov;31(4):337-44. Oncogene amplification in medulloblastoma: analysis of a case by comparative genomic hybridization and fluorescence in situ hybridization. Jay V, Squire J, Bayani J, Alkhani AM, Rutka JT, Zielenska M. Division of Pathology, Hospital for Sick Children-University of Toronto, Ontario, Canada. vjay@sickkids.on.ca We describe amplification of the MYCC oncogene in a medulloblastoma with aggressive clinical behavior. The patient was a six year old boy who underwent gross total surgical excision of a cerebellar tumor. Despite chemotherapy and total neuraxis radiation, the clinical course was one of relentless progression, with extensive subarachnoid spread and death within eight months of presentation. The pathological features were consistent with the recently described, "large cell variant" of medulloblastoma. Tumor cells exhibited large vesicular nuclei, prominent nucleoli and strong immunoreactivity for synaptophysin. Polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) assay revealed no evidence of MYCN amplification or 1p deletion in the tumor. FISH analysis revealed evidence of MYCC amplification in the 20- to 30-fold range. Comparative genomic hybridization (CGH) revealed regions of gains and amplification in three locations, with gains of chromosome 7, amplification of 8q24 (corresponding to the MYCC locus) and gains of the long arm of chromosome 17 (suggestive of isochromosome 17q). While conventional karyotypic analysis was not successful in the present case, CGH provided invaluable information about gene amplification and losses/gains of chromosomes and chromosomal regions. Thus, CGH is a powerful technique applicable to frozen or paraffin-embedded material which helps to ascertain the presence of gene amplification even without prior knowledge of the gene to be tested. Publication Types: Case Reports PMID: 10643003 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 146: J Clin Oncol. 2000 Jan;18(1):18-26. Biologic variables in the outcome of stages I and II neuroblastoma treated with surgery as primary therapy: a children's cancer group study. Perez CA, Matthay KK, Atkinson JB, Seeger RC, Shimada H, Haase GM, Stram DO, Gerbing RB, Lukens JN. Department of Surgery, University of Southern California School of Medicine and Children's Hospital, Los Angeles, USA. PURPOSE: To determine prospectively whether surgery alone is sufficient therapy for Evans stages I and II neuroblastoma and to define biologic and clinical features having prognostic potential for this group. PATIENTS AND METHODS: Between June 1989 and August 1995, 374 eligible children (age range, 0 to 18 years) with newly diagnosed stage I (n = 141) and stage II (n = 233) neuroblastoma were registered onto Children's Cancer Group trial 3881. Surgical resection was the only primary therapy except in cases with spinal cord compression, where radiation therapy was allowed. Event-free survival (EFS) and overall survival (OS) were analyzed by life-table methods according to clinical and biologic features. RESULTS: EFS and OS (mean +/- SE) for all stage I patients were 93% +/- 3.0% and 99% +/- 1.0%, respectively, compared with 81% +/- 4.0% and 98% +/- 2. 0%, respectively, for stage II patients. The significantly higher recurrence rate among stage II patients was managed successfully in 38 of 43 children with either surgery or multimodality treatment. There was one death among stage I patients and six among stage II. For stage II patients tumor MYCN gene amplication, unfavorable histopathology, an age greater than 2 years, and positive lymph nodes predicted a lower OS (P <.05). CONCLUSION: Children with stages I and II neuroblastoma have 98% survival with surgery alone as primary therapy. Supplemental treatment was necessary in only 10% of stage I patients and 20% of stage II patients. In children with localized neuroblastoma, a subset of patients that are at higher risk for death can be defined as those with stage II disease who have tumor MYCN amplification or who are >/= 2 years of age with either unfavorable histopathology or positive lymph nodes. Publication Types: Clinical Trial Multicenter Study PMID: 10623689 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 147: Cancer Genet Cytogenet. 2000 Jan 1;116(1):87-8. Expulsion of amplified MYCN from neuroblastoma tumor cells. Freeman-Edward J, O'Neill S, Lastowska M, Bown N. Publication Types: Letter PMID: 10616541 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 148: Genes Chromosomes Cancer. 2000 Feb;27(2):143-52. An integrated 5-Mb physical, genetic, and radiation hybrid map of a 1p36.1 region implicated in neuroblastoma pathogenesis. Spieker N, Beitsma M, van Sluis P, Roobeek I, den Dunnen JT, Speleman F, Caron H, Versteeg R. Department of Human Genetics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Common genetic aberrations of neuroblastoma are deletions of the short arm of chromosome 1 (1p36) and MYCN amplification. Our deletion analysis of 25 tumor cell lines and 171 tumors strongly suggests that 1p harbors several tumor suppressor loci. Distinct loci are involved in MYCN single-copy versus MYCN-amplified neuroblastoma. Deletions in MYCN single-copy tumors have a shortest region of overlap (SRO) of 20 cM at 1p36.3. MYCN-amplified tumors have large deletions with an SRO of about 60 cM, from 1p36.1 to the telomere. This SRO is defined by D1S7 (1p36.1), which was the most distal locus retained. Therefore, a suppressor gene associated with MYCN-amplified tumors probably maps within a few megabases distal of D1S7. In order to map this locus, we further refined this SRO. We mapped the breakpoint of the MYCN-amplified neuroblastoma with the smallest 1p deletion between 56.6 and 57.2 cM from 1pter. Pulsed-field gel electrophoresis and radiation hybrid mapping were used to construct a 5-Mb physical map of this region. The map includes the region from 82.73 till 92.89 cR from 1pter. About half of it was isolated in P1 and PAC clones. The region harbors the genes FGR, SLC9A1, HMG17, EXTL1, AML2, RH, OP18, four ESTs, and a newly identified gene with a transcript size of approximately 7 Kb. Several of the mapped genes have a putative role in cell growth, differentiation, and morphogenesis. Genes Chromosomes Cancer 27:143-152, 2000. Copyright 2000 Wiley-Liss, Inc. PMID: 10612802 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 149: Oncogene. 1999 Nov 18;18(48):6810-7. Nmi protein interacts with regions that differ between MycN and Myc and is localized in the cytoplasm of neuroblastoma cells in contrast to nuclear MycN. Bannasch D, Weis I, Schwab M. Division of Cytogenetics-H0400, Deutsches Krebsforschungszentrum, Heidelberg, Germany. Myc family proteins play an important role in cellular processes such as proliferation, differentiation, apoptosis and transformation. A number of interaction partners of Myc have been identified, such as Max, p107, TBP, YY1, Miz-1, AP-2 and Nmi. Both Max and Nmi also bind to MycN. In contrast to the well defined binding of Max to Myc family proteins the interaction of Nmi with Myc or MycN is only poorly characterized. By employing the yeast two-hybrid system we have mapped the regions of MycN and Myc responsible for binding to Nmi. For MycN exclusively a central region mediates binding to Nmi. In contrast, for Myc a C-terminal portion of the protein, and possibly also a central part, is involved in Nmi interaction. Nmi does not interact with Max and has no transactivation capabilities in yeast, suggesting that Nmi alone is not a transcriptional activator in mammalian cells. Immunofluorescence demonstrates that both in 293 embryonic kidney cells and in Kelly neuroblastoma cells all detectable ectopically expressed Nmi is localized in the cytoplasm, in part in a punctate, granular pattern. MycN, which is highly expressed in Kelly cells consequent to amplification, appears to be localized exclusively in the nuclei. This directly demonstrates that in the same cell at least the major proportion of MycN and Nmi is localized in different cellular compartments. This result is confirmed by the finding that endogenous Nmi, which is expressed in Kelly cells only after stimulation with interferon gamma, is detected exclusively in the cytoplasm of these cells. Therefore only a very small amount of MycN and Nmi is likely to be involved in MycN/Nmi interaction in vivo. PMID: 10597290 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 150: J Clin Oncol. 1999 Jul;17(7):2264-79. Molecular biology of neuroblastoma. Maris JM, Matthay KK. Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, PA 19104-4318, USA. maris@email.chop.edu PURPOSE AND RESULTS: Neuroblastoma, the most common solid extracranial neoplasm in children, is remarkable for its clinical heterogeneity. Complex patterns of genetic abnormalities interact to determine the clinical phenotype. The molecular biology of neuroblastoma is characterized by somatically acquired genetic events that lead to gene overexpression (oncogenes), gene inactivation (tumor suppressor genes), or alterations in gene expression. Amplification of the MYCN proto-oncogene occurs in 20% to 25% of neuroblastomas and is a reliable marker of aggressive clinical behavior. No other oncogene has been shown to be consistently mutated or overexpressed in neuroblastoma, although unbalanced translocations resulting in gain of genetic material from chromosome bands 17q23-qter have been identified in more than 50% of primary tumors. Some children have an inherited predisposition to develop neuroblastoma, but a familial neuroblastoma susceptibility gene has not yet been localized. Consistent areas of chromosomal loss, including chromosome band 1p36 in 30% to 35% of primary tumors, 11q23 in 44%, and 14q23-qter in 22%, may identify the location of neuroblastoma suppressor genes. Alterations in the expression of the neurotrophins and their receptors correlate with clinical behavior and may reflect the degree of neuroblastic differentiation before malignant transformation. Alterations in the expression of genes that regulate apoptosis also correlate with neuroblastoma behavior and may help to explain the phenomenon of spontaneous regression observed in a well-defined subset of patients. CONCLUSION: The molecular biology of neuroblastoma has led to a combined clinical and biologic risk stratification. Future advances may lead to more specific treatment strategies for children with neuroblastoma. Publication Types: Review Review, Tutorial PMID: 10561284 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 151: Am J Pathol. 1999 Nov;155(5):1439-43. MYCN gene amplification. Identification of cell populations containing double minutes and homogeneously staining regions in neuroblastoma tumors. Yoshimoto M, Caminada De Toledo SR, Monteiro Caran EM, de Seixas MT, de Martino Lee ML, de Campos Vieira Abib S, Vianna SM, Schettini ST, Anderson Duffles Andrade J. Division of Genetics, Department of Morphology, Universidade Federal de Sao Paulo, Escola Paulista de Medicina, Sao Paulo, Brazil. Neuroblastoma is the second most common solid tumor occurring in children. Amplification of the MYCN oncogene is associated with poor prognosis. To identify neuroblastoma tumors with MYCN amplification, we studied the number of copies of MYCN in interphase cells by fluorescence in situ hybridization in 20 neuroblastoma patients. MYCN amplification appeared in 7 tumor specimens. Interphase and metaphase studies showed a tumor cell population with both forms of amplification, double minutes and homogeneously staining regions, in two patients. These patients showed a smaller tumor cell subpopulation with the presence of more than one homogeneously staining region, suggesting that gene amplification was undergoing karyotype evolution. PMID: 10550298 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 152: Clin Chem. 1999 Nov;45(11):1918-24. Real-time quantitative PCR for the measurement of MYCN amplification in human neuroblastoma with the TaqMan detection system. Raggi CC, Bagnoni ML, Tonini GP, Maggi M, Vona G, Pinzani P, Mazzocco K, De Bernardi B, Pazzagli M, Orlando C. Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, 50139 Florence, Italy. BACKGROUND: Neuroblastoma is the most common extracranial malignant solid tumor in children under 5 years and is characterized by a wide clinical and biological heterogeneity, from spontaneously regressive forms to cancers with a rapid and fatal progression. MYCN oncogene amplification is considered the most important prognostic factor to evaluate survival and therapeutic choices in these patients. METHODS: Here we present a new assay for rapid and accurate measurement of MYCN amplification, based on real-time quantitative PCR with the TaqMan(TM) reaction. The degree of MYCN amplification was derived from the ratio of the MYCN oncogene and the single-copy reference gene, beta-actin. The absolute abundance of these two genes in tumor sample DNA was obtained by extrapolation on external calibration curves generated with reference DNA. RESULTS: We found a variable degree of MYCN amplification, from 2 to 29, in 26 of 49 (53%) neuroblastomas. These results were well correlated to those obtained with a competitive PCR assay in the same samples (r = 0. 987). MYCN amplification was associated mainly with advanced cancer stages, and the analysis of overall survival confirmed that the measurement of MYCN amplification is a predictor of patient outcome in neuroblastoma. Patients without MYCN amplification had a cumulative survival significantly higher than patients with low (<9; P = 0.02) and high (>/=9; P = 0.03) oncogene amplification. CONCLUSION: The assay is rapid and reproducible and does not require any post-PCR analytical procedure. PMID: 10545060 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 153: J Clin Oncol. 1999 Oct;17(10):3216-20. Long-term survivors of advanced neuroblastoma with MYCN amplification: A report of 19 patients surviving disease-free for more than 66 months. Kawa K, Ohnuma N, Kaneko M, Yamamoto K, Etoh T, Mugishima H, Ohhira M, Yokoyama J, Bessho F, Honna T, Yoshizawa J, Nakada K, Iwafuchi M, Nozaki T, Mimaya J, Sawada T, Nakamura T, Miyata H, Yamato K, Tsuchida Y. Study Group of Japan for Treatment of Advanced Neuroblastoma, Izumi, Japan. PURPOSE: According to initial reports, stage 4 neuroblastoma patients with amplification of the MYCN proto-oncogene developed progressive disease within 8 months. The prognosis for such patients, however, should now be reevaluated in light of recent results achieved with up-to-date combination chemotherapy. PATIENTS AND METHODS: Patients with stage 3, 4, and 4S neuroblastoma and more than 10 copies of MYCN received induction chemotherapy, which from January 1985 to February 1991 consisted of regimen A(1 )(cyclophosphamide 1,200 mg/m(2) on day 1, vincristine 1.5 mg/m(2) on day 1, pirarubicin 40 mg/m(2) on day 3, and cisplatin 90 mg/m(2) on day 5) and from March 1991 to September 1993 consisted of regimen A(3 )(cyclophosphamide 1,200 mg/m(2) on days 1 and 2, pirarubicin 40 mg/m(2) on day 3, etoposide 100 mg/m(2) on days 1 through 5, and continuous infusion cisplatin 25 mg/m(2) on days 1 through 5). Most of these patients underwent radical surgery to remove the original tumor and local metastases, irradiation, and supralethal preconditioning regimens, followed by blood stem-cell transplantation (SCT). Data on the patients were collected in December 1998, and the factors contributing to disease-free survival were analyzed. RESULTS: During the study period, 66 patients with more than 10 copies of MYCN were treated. Five of nine patients with stage 3 disease, 13 of 55 with stage 4, and one of two with stage 4S survived for at least 66 months. It is interesting that all but one patient who survived for more than 66 months underwent SCT, in contrast with only five of 45 patients who died. CONCLUSION: Not all patients with advanced neuroblastoma who have more than 10 copies of MYCN will die. The requisites for survival in such patients seem to be intensive induction chemotherapy, effective surgery, irradiation, and the use of SCT. PMID: 10506621 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 154: Oncogene. 1999 Sep 2;18(35):4948-57. Allelic deletion at 11q23 is common in MYCN single copy neuroblastomas. Guo C, White PS, Weiss MJ, Hogarty MD, Thompson PM, Stram DO, Gerbing R, Matthay KK, Seeger RC, Brodeur GM, Maris JM. Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, PA 19104, USA. Deletions of the long arm of chromosome 11 (11q) have been noted in primary neuroblastomas, but a comprehensive analysis has not been performed. Therefore, we analysed 331 neuroblastomas (295 sporadic, 15 familial and 21 tumor-derived cell lines) to determine the prevalence of 11q allelic deletions, to map the location of a putative tumor suppressor gene and to perform clinical correlative studies. Assays for loss of heterozygosity (LOH) were performed at 24 microsatellite loci spanning 11q. LOH was observed at multiple 11q loci in 129/295 (44%) sporadic neuroblastomas, 5/15 (33%) familial neuroblastomas, and 5/21 (24%) neuroblastoma cell lines. A single region of 2.1 cM within 11q23.3, flanked by markers D11S1340 and D11S1299, was deleted in all specimens with 11q LOH. Allelic loss at 11q23 was inversely related to MYCN amplification (P<0.001). Within the subset of cases with a single copy of MYCN, 11q LOH was associated with advanced stage disease (P=0.008), unfavorable histopathology (P=0.042), and decreased overall survival probability (P=0.008). However, 11q LOH was not independently prognostic in multivariate analyses. These data support the hypothesis that a tumor suppressor gene mapping within 11q23.3 is commonly inactivated during the malignant evolution of a large subset of neuroblastomas, especially those with unamplified MYCN. PMID: 10490829 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 155: Eur J Biochem. 1999 Aug;263(3):757-64. Down-regulation of endothelial cell growth inhibitors by enhanced MYCN oncogene expression in human neuroblastoma cells. Fotsis T, Breit S, Lutz W, Rossler J, Hatzi E, Schwab M, Schweigerer L. Division of Hematology, Children's University Hospital, Ruprecht- Karls-Universitat, INF 150, Heidelberg, Germany. thfotsis@cc.uoi.gr Recent evidence indicates that the genetic alterations of the multistage process of malignant transformation appear to activate tumor neovascularization by altering the balance between stimulators and inhibitors of angiogenesis. In the present study, we have attempted to define the effect of enhanced MYCN oncogene expression on the profile of endothelial cell growth modulators in neuroblastoma cells. We report here that conditioned medium of human neuroblastoma cells with normal MYCN expression contains three inhibitors of endothelial cell proliferation, which appear to be novel proteins as judged by their physicochemical, immunological and biological properties. All three inhibitors are diminished or become undetectable upon experimental increase of MYCN expression. Our results suggest that enhanced MYCN expression in human neuroblastoma cells alters the angiogenic balance by down-regulating endothelial cell growth inhibitors but leaving the expression of the stimulators unaffected. These data shed light on the molecular mechanisms linking the genetic changes of malignant transformation with initiation of tumor angiogenesis. Moreover, our observations might explain the poor prognosis of human neuroblastomas following MYCN oncogene amplification through initiation of angiogenesis and subsequent tumor growth and spread. PMID: 10469139 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 156: Cancer Res. 1999 Jul 15;59(14):3365-8. Expression of B-myb in neuroblastoma tumors is a poor prognostic factor independent from MYCN amplification. Raschella G, Cesi V, Amendola R, Negroni A, Tanno B, Altavista P, Tonini GP, De Bernardi B, Calabretta B. ENEA CR Casaccia, Section of Toxicology and Biomedical Sciences, Rome, Italy. The transcription factors of the Myb family are expressed in several tissues and play an important role in cell proliferation, differentiation, and survival In this study, the expression of A-myb, B-myb, and c-myb was investigated in a group of 64 neuroblastomas at different dinical stages by a sensitive reverse transcription-PCR tchnique and correlated with patients' survival. All of the myb genes were frequently expressed in neuroblastoma tumors. Interestingly, the expression of B-myb, which was detected in 33 cases, was associated with an increased risk of death (P = 0.027 in a univariate analysis), whereas there was no correlation with A-myb and c-myb expression. In addition, in a multivariate Cox regression analysis that included myb gene expression, MYCN status, age at diagnosis, and tumor staging, MYCN amplification and B-myb expression were independently associated to an increased risk (P < 0.01 and P = 0.015, respectively). In overall survival curves obtained by stratifying the neuroblastoma cases on the basis of MYCN status and B-myb expression, the group of patients without MYCN amplification and positive for B-myb expression had worse survival probability than that without MYCN amplification and nonexpressing B-myb (P < 0.01). In summary, these findings provide the first demonstration that B-myb expression can be a useful prognostic marker in human neuroblastoma. Moreover, B-myb expression has a prognostic value complementary to MYCN amplification and can identify a group of high-risk patients that would not be predicted on the basis of the MYCN status only. PMID: 10416595 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 157: Pediatr Radiol. 1999 Jul;29(7):497-502. Neuroblastoma arising from the organ of Zuckerkandl: an unusual site with a favorable biologic outcome. Berdon WE, Stylianos S, Ruzal-Shapiro C, Hoffer F, Cohen M. Department of Radiology, Division of Pediatric Radiology, Babies & Children's Hospital of New York, 3959 Broadway, BHN 3-318, New York, NY 10032, USA. BACKGROUND: Prognosis in neuroblastoma has been shown to correlate with age and stage at diagnosis and site of origin. Extra-abdominal tumors (chest, neck, pelvis) do better in terms of survival than tumors arising from the upper abdomen. OBJECTIVE: We evaluated a subgroup of abdominal neuroblastomas arising near to the aortic bifurcation (commonly called organ of Zuckerkandl, O. Z.) to assess their biologic outcome and problems in diagnosis and therapy. MATERIALS AND METHODS: Sixteen O. Z. primary tumors were seen at three children's hospitals. Their clinical records and imaging studies were reviewed, including the sonographic, CT, and MRI findings. When available, MYCN amplification was noted (MYCN is the current term previously called N-MYC). RESULTS: Despite more than half of the tumors being very large, survival was the rule, with only one fatality (following multiple local recurrences). Only one patient (who survived) had bone metastases. The larger masses were usually palpated in otherwise well children, while the smaller ones were found in the course of evaluation for unrelated problems such as urinary tract infection. Intraspinal extension was common, though usually asymptomatic. MYCN amplification was absent in the four patients studied. CONCLUSIONS: Lower abdominal (O. Z.) neuroblastomas present technical problems of surgical removal, but form a group with a favorable outcome similar to cervical and thoracic primary sites. MRI was useful in delineating intraspinal extension. PMID: 10398782 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 158: Clin Cancer Res. 1999 Jun;5(6):1491-6. High-level expression of EPHB6, EFNB2, and EFNB3 is associated with low tumor stage and high TrkA expression in human neuroblastomas. Tang XX, Evans AE, Zhao H, Cnaan A, London W, Cohn SL, Brodeur GM, Ikegaki N. Division of Oncology, The Children's Hospital of Philadelphia, Abramson Research Center, Pennsylvania 19104-4318, USA. Neuroblastoma (NB) is a common pediatric tumor of neural crest origin that is biologically and clinically heterogeneous. EPH family receptor tyrosine kinases and ephrin ligands play fundamental roles in neurodevelopmental processes. Recently, we found that NB cell lines expressed several EPHB and EFNB transcripts, which encode EPHB subgroup receptors and ephrin-B subgroup ligands, respectively. To explore the role of EPHB receptors and ephrin-B ligands in the biology of NB, we examined the expression of EPHB and EFNB transcripts in 47 primary NB specimens. Multiple EPHB and EFNB transcripts were expressed in all of the NB tumors examined, suggesting the involvement of these transcripts in modulating the biological behavior of NB. Higher levels of EPHB6, EFNB2, and EFNB3 expression were found in low-stage tumors (stage 1, 2, and 4S) than in advanced-stage tumors (stage 3 and 4; P = 0.0013, P = 0.0048, and P = 0.027, respectively). Expression of TrkA, a well-established prognostic marker of favorable NB, was positively correlated with EPHB6, EFNB2, and EFNB3 expression (P < 0.0001, P = 0.0019, and P = 0.0001, respectively). MYCN-amplified tumors expressed lower levels of EPHB6, EFNB2, EFNB3, and TrkA transcripts compared to nonamplified tumors (P = 0.0006, P = 0.0023, P = 0.0048, and P = 0.0001, respectively). These data suggest that high-level expression of EPHB6, EFNB2, and EFNB3 is associated with favorable NB and that low-level expression of EPHB6, EFNB2, and EFNB3 correlates with aggressive MYCN-amplified NB. Thus, EPHB6, EFNB2, and EFNB3 may have biological relevance in NB. Further investigation on the biology of these genes may help provide insight into the treatment of NB. PMID: 10389937 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 159: Oncol Rep. 1999 Jul-Aug;6(4):891-6. Clinical relevance of molecular markers in neuroblastoma: results from a single institution. Gallego S, Parareda A, Munell F, Sanchez de Toledo J, Reventos J. Unitat d'Oncologia Pediatrica, Hospital Materno-Infantil Vall d'Hebron, 08035 Barcelona, Spain. Neuroblastomas, the most common extracranial solid tumors in children, present an extremely heterogeneous behaviour that can be explained in part by their genetic abnormalities. Thirty-four patients treated at the Pediatric Oncology Unit, Hospital Vall d'Hebron from 1993 to 1997 were prospectively studied to determine the relative prognostic impact of a number of clinical and molecular factors. The factors studied were: ploidy, MYCN and 1p status, and TRK-A expression, in addition to age, stage and histology. Their impact on prognosis was analyzed. In univariate analysis, advanced stage, unfavorable histology, diploidy, MYCN amplification, and 1p deletion were identified as adverse prognostic factors; TRK-A expression was associated with favorable prognosis. After multivariate analysis, only MYCN amplification proved to be an independent adverse prognostic factor (p=0.03), whereas TRK-A expression identified a subset of good-prognosis patients (p=0.003). PMID: 10373677 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 160: J Pediatr Hematol Oncol. 1999 May-Jun;21(3):181-9. Comment in: J Pediatr Hematol Oncol. 1999 May-Jun;21(3):178-80. Metastatic sites in stage IV and IVS neuroblastoma correlate with age, tumor biology, and survival. DuBois SG, Kalika Y, Lukens JN, Brodeur GM, Seeger RC, Atkinson JB, Haase GM, Black CT, Perez C, Shimada H, Gerbing R, Stram DO, Matthay KK. Department of Pediatrics, University of California School of Medicine, San Francisco, USA. PURPOSE: The goal of this study was to determine the incidence of metastatic sites in neuroblastoma and the extent to which metastatic sites correlate with age, tumor biology, and survival. PATIENTS AND METHODS: All 648 patients with stage IV and IVS neuroblastoma registered on Children's Cancer Group protocols 3881 and 3891 were analyzed. Metastatic site data were provided by treating institutions and reviewed in patients with central nervous system (CNS), intracranial, lung, or "other" metastases. RESULTS: The incidence of metastatic sites at diagnosis was 70.5% in bone marrow, 55.7% in bone, 30.9% in lymph nodes, 29.6% in liver, 18.2% in intracranial and orbital sites, 3.3% in lung, and 0.6% in CNS. Event-free survival (EFS) was decreased in patients with bone, bone marrow, CNS, intracranial/ orbital, lung, and pleural metastases, and improved in those with liver and skin metastases. In infants, MYCN amplification and unfavorable Shimada histopathology correlated with increased frequencies of bone and intracranial or orbital metastases. In older patients, MYCN amplification correlated with increased frequencies of intracranial or orbital, liver, and lung metastases. Multivariate analysis revealed that metastatic site is not an independent prognostic factor. CONCLUSIONS: Metastatic pattern in neuroblastoma differs with age and correlates with tumor biological features and EFS. These correlations could reflect changes in host or tumor biological features with age resulting in differences in metastatic capacity or tumor affinity for specific sites. PMID: 10363850 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 161: Int J Mol Med. 1999 Jun;3(6):585-9. Variable expression and absence of mutations in p73 in primary neuroblastoma tumors argues against a role in neuroblastoma development. Ejeskar K, Sjoberg RM, Kogner P, Martinsson T. Department of Clinical Genetics, University of Gothenburg, Sahlgrenska University Hospital/East, S-416 85 Gothenburg, Sweden. In neuroblastoma, a childhood tumor of neural crest, a tumor suppressor gene located at 1p36 has been implicated to play a major role in tumor aggressiveness and clinical prognosis. We have examined 30 different staged primary neuroblastoma tumors using RT-PCR, for expression of the p73 gene located at 1p36.3, and its correlation to other clinical and biological features of these tumors. No correlation between expression of p73 and MYCN-amplification or 1p-deletion could be found, five of ten 1p-deleted tumors showed detectable levels of p73, and no mutations could be detected, neither in the retained alleles nor in any other parts of the material. In five 1p-deleted cases the origin of deletion were determined, two were of maternal and three of paternal origin. Both tumors with maternal 1p-loss showed detectable levels of p73, whereas the three with paternal loss did not. This suggests that p73 is expressed from the paternal allele only in advanced staged neuroblastoma tumors. Furthermore, it suggests absence of correlation between p73-expression and stage in these tumors. In conclusion, we could find no evidence for p73 being the neuroblastoma tumor suppressor gene in 1p36. PMID: 10341287 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 162: Pathol Res Pract. 1999;195(4):237-42. Establishment and characterization of a serous papillary adenocarcinoma cell line of the human ovary in a serum-free culture. Emoto M, Oshima K, Ishiguro M, Iwasaki H, Kawarabayashi T, Kikuchi M. Department of Obstetrics and Gynecology, Fukuoka University School of Medicine, Japan. In order to clarify the biologic characteristics of serous papillary adenocarcinoma of the human ovary, we tried to establish a continuous cell line using four primary tumor specimens derived from four patients with such tumors. We also evaluated c-myc, MYCN and c-erbB2 gene amplification in the cultured cells, and the xenografts, as well as in these four primary tissue specimens by a Southern blot analysis. One continuous cell line, named as FU-OV-1, was established in a serum-free culture system and this line propagated continuously for 96 serial passages over a 2-year-period in vitro. FU-OV-1 reproduced and still maintained the characteristics of the original tumor. C-myc gene amplification was found in the FU-OV-1 cells, and the xenografts, as well as in only the primary tumor of FU-OV-1 out of the four primary serous papillary adenocarcinomas. However, neither MYCN amplification nor c-erbB2 amplification was observed in any tumor specimens including FU-OV-1 cells. In conclusion, FU-OV-1 is thus considered to be a useful system for studying the biological behavior of serous papillary adenocarcinoma in the human ovary. The results of this study suggest that c-myc gene amplification might thus be associated with the progression of this tumor both in vitro and in vivo. PMID: 10337661 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 163: Biochem Mol Biol Int. 1999 Apr;47(4):563-8. Expression of two dead box genes (DDX1 and DDX6) is independent of that of MYCN in human neuroblastoma cell lines. Akiyama K, Akao Y, Yokoyama M, Nakagawa Y, Noguchi T, Yagi K, Nishi Y. Pharmaceutical Frontiers Research Laboratories, Japan Tobacco, Inc., Yokohama. To examine whether two DEAD box genes, DDX1 and DDX6, would have some roles in the progression of tumors, we investigated the correlation of the expression of these genes with that of MYCN in neuroblastomas either with or without MYCN amplification. The mRNA of MYCN was observed only in the cell lines with amplification of MYCN. The mRNAs of DDX1 and DDX6 were found in all the cell lines examined, but the correlation between the mRNA levels of DDX1 or DDX6 and MYCN was poor. These findings suggest that the expression of neither DEAD box gene is correlated with the gene expression of MYCN. PMID: 10319407 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 164: Clin Cancer Res. 1999 Mar;5(3):601-9. Rapid detection of MYCN gene amplification and telomerase expression in neuroblastoma. Hiyama E, Hiyama K, Yokoyama T, Fukuba I, Yamaoka H, Shay JW, Matsuura Y. Department of General Medicine, Hiroshima University School of Medicine, Hiroshima, Japan. eiso@mcai.med.hiroshima-u.ac.jp Amplification of the MYCN gene and high telomerase activity predict a poor prognosis for the patients with neuroblastoma. We used PCR techniques for rapid detection of MYCN gene amplification and human telomerase reverse transcriptase (hTERT) expression in neuroblastoma specimens. The detection of MYCN gene amplification is based on differential PCR in which three primer pairs were used to coamplify a 178-bp fragment of target MYCN gene with two reference gene fragments, a 237-bp of p53 exon 7 and a 120-bp of beta-globin exon 3, in a single tube of 40 surgically resected tumor samples. MYCN amplification was identified by this differential PCR in all 10 samples carrying more than 10 copies (already known to have MYCN gene amplification by Southern blot analysis). There were no false-negative or false-positive cases, and the relative intensity of MYCN bands in the differential PCR correlated significantly with the copy number determined by Southern blot analysis (y = 0.99, P<0.0001). This protocol was also applicable in the biopsy or aspirated samples, as well as the paraffin-embedded tissues, and in detecting intratumoral heterogeneity. Using RT-PCR procedures, hTERT mRNA expression was detectable in all 13 tumors with high telomerase activity. These nonradioisotopic PCR-based protocols for detecting MYCN gene amplification and hTERT mRNA expression are rapid and reliable and are likely to be useful to determine the biological behavior of neuroblastoma. PMID: 10100712 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 165: Naturwissenschaften. 1999 Feb;86(2):71-8. Human neuroblastoma: from basic science to clinical debut of cellular oncogenes. Schwab M. Deutsches Krebsforschungszentrum, Abteilung Zytogenetik H0400, Heidelberg, Germany. m.schwab@dkfz-heidelberg.de Neuroblastoma is a childhood embryonic tumor of migrating neuroectodermal cells derived from the neural crest and destined for the adrenal medulla and the sympathetic nervous system. It very often has a rapidly progressive clinical course, and although many advances have been made in understanding the development of this tumor, improving the survival rates particularly in patients with metastatic tumor has been a frustrating experience. The mechanisms leading to neuroblastoma are largely unclear, but nonrandom chromosomal changes discovered early suggested the involvement of genetic alterations. Most prominent among these is the amplification of the oncogene MYCN, which identifies a group of patients who have a particularly dire prognosis. Amplified MYCN is used today as a prognostic marker on which therapy design is based to a large extent. An unusual aspect of neuroblastoma is the high rate at which tumors regress spontaneously, even in infants with extensive liver involvement and numerous subcutaneous nodules. Identifying the molecular and cellular basis of spontaneous regression could result in improved therapeutic approaches. Neuroblastoma is a model tumor with many fascinating aspects but has remained a challenge to the pediatric oncologist. Publication Types: Review Review, Tutorial PMID: 10084150 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 166: Oncogene. 1999 Feb 18;18(7):1479-86. MycN sensitizes neuroblastoma cells for drug-induced apoptosis. Fulda S, Lutz W, Schwab M, Debatin KM. University Children's Hospital, Ulm, Germany. Amplification of the MYCN gene is found in a large proportion of neuroblastoma and considered as an adverse prognostic factor. To investigate the effect of ectopic MycN expression on the susceptibility of neuroblastoma cells to cytotoxic drugs we used a human neuroblastoma cell line harboring tetracycline-controlled expression of MycN. Neither conditional expression of MycN alone nor low drug concentrations triggered apoptosis. However, when acting in concert, MycN and cytotoxic drugs efficiently induced cell death. Apoptosis depended on mitochondrial permeability transition and activation of caspases, since the mitochondrion-specific inhibitor bongkrekic acid and the caspase inhibitor zVAD-fmk almost completely abrogated apoptosis. Loss of mitochondrial transmembrane potential and release of cytochrome c from mitochondria preceded activation of caspase-8 and caspase-3 and cleavage of PARP. CD95 expression was upregulated by treatment with cytotoxic drugs, while MycN cooperated with cytotoxic drugs to increase sensitivity to CD95-induced apoptosis and enhancing CD95-L expression. MycN overexpression and cytotoxic drugs also synergized to induce p53 and Bax protein expression, while Bcl-2 and Bcl-X(L) protein levels remained unchanged. Since amplification of MYCN is usually associated with a poor prognosis, these findings suggest that dysfunctions in apoptosis pathways may be a mechanism by which MycN-induced apoptosis of neuroblastoma cells is inhibited. PMID: 10050884 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 167: Int J Cancer. 1999 Jan 5;80(1):54-9. Disomy 1 with terminal 1p deletion is frequent in mass-screening-negative/late-presenting neuroblastomas in young children, but not in mass-screening-positive neuroblastomas in infants. Kaneko Y, Kobayashi H, Maseki N, Nakagawara A, Sakurai M. Department of Cancer Chemotherapy, Saitama Cancer Center Hospital, Ina, Japan. kaneko@saitama-cc.go.jp The mass screening (MS) of neuroblastoma has been undertaken in Japan by measuring urinary catecholamine metabolites in infants at the age of 6 months. To clarify the biological characteristics of MS-positive (MS+) tumors in infants and MS-negative (MS-)/late-presenting tumors in young children, metaphase cytogenetic and/or interphase 2-color FISH analyses using terminal 1p and pericentromeric 1q probes were performed on 246 (186 MS+ and 60 MS-) patients with neuroblastomas. The 246 tumors were classified into 4 groups on the basis of the constitution of chromosome 1; 22 tumors had disomy 1 with no 1p deletion (Dis1Norm1p); 41 tumors had disomy 1 or tetrasomy 1, all with the 1p deletion (Dis1Del1p); 164 tumors had trisomy 1, pentasomy 1, or a mixed population of cells with trisomy 1 and cells with tetrasomy 1, none with 1p deletion (Tris1Norm1p); 19 tumors with the same copy numbers of chromosome 1 as the Tris1Norm1p group, had 1p deletion (Tris1Del1p). mycn amplification was absent in the Dis1Norm1p and Tris1Del1p groups, frequent in the Dis1Del1p group (24/41), and rare in the Tris1Norm1p group (3/164) (p < 0.0001). Event-free survival at 5 years was lowest [19.5%; 95% confidence interval (CI), 5.1-33.9] in the Dis1Del1p group, highest in the Tris1Norm1p (96.3%; 95% CI, 93.5-99.2) and Tris1Del1p (94.7%; 95% CI, 84.7-104.8) groups, and intermediate but varied (54.5%; 95% CI, 33.7-75.4) in the Dis1Norm1p group (p < 0.0001). Of the MS+ tumors, 90% were Tris1Norm1p or Tris1Del1p, and 55% of the MS- tumors were Dis1Del1p. The finding that the Dis1Del1p tumors were frequent in MS- but not in MS+ tumors suggests the limited efficacy of the MS program into reducing mortality from neuroblastoma. PMID: 9935230 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 168: Int J Cancer. 1999 Feb 9;80(4):630-1. Wild-type sequence of MYCN in neuroblastoma cell lines. Hogarty MD, Brodeur GM. Publication Types: Letter PMID: 9935168 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 169: Exp Cell Res. 1999 Jan 10;246(1):183-92. Depletion of glutathione by buthionine sulfoxine is cytotoxic for human neuroblastoma cell lines via apoptosis. Anderson CP, Tsai JM, Meek WE, Liu RM, Tang Y, Forman HJ, Reynolds CP. Division of Hematology-Oncology, Childrens Hospital Los Angeles, 4650 Sunset Boulevard, Los Angeles, California, 90027, USA. cpreynol@hsc.usc.edu Buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis and has been used to sensitize tumor cells to alkylating agents, but has minimal single-agent cytotoxicity for most cell types. We determined the cytotoxicity of BSO for 18 (12 MYCN amplified; 6 MYCN nonamplified) human neuroblastoma cell lines using DIMSCAN, a digital image microscopy cytotoxicity assay. D-L(R:S) BSO was highly cytotoxic (>3 logs of cell kill) for most neuroblastoma cell lines, with 17/18 cell lines having IC90 values (range 2. 1->1000 microM) below equivalent steady state plasma levels of L(R:S) BSO reported in adult human trials. Cell lines with genomic amplification of MYCN were more sensitive to BSO than MYCN nonamplified cell lines (P = 0.04). D-L(R:S) BSO (500 microM for 72 h) induced apoptosis as detected by DNA laddering, nuclear morphology, and TUNEL staining of DNA fragments using flow cytometry. Maximal cell killing occurred within 48 h and was antagonized byic value in neuroblastoma. Copyright 1999 Academic Press. PMID: 9882527 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 170: Mod Pathol. 1998 Dec;11(12):1222-7. N-myc gene amplification in rhabdomyosarcoma detected by fluorescence in situ hybridization: its correlation with histologic features. Hachitanda Y, Toyoshima S, Akazawa K, Tsuneyoshi M. Second Department of Pathology, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Fluorescence in situ hybridization (FISH) was applied to 15 alveolar rhabdomyosarcomas (A-RMSs) and 14 embryonal RMSs (E-RMSs) to detect N-myc (also called MYCN) oncogene amplification. The results were compared with histologic characteristics and clinical factors. The number of surviving patients in each subtype was 5 of 15 with A-RMS and 5 of 14 with E-RMS. N-myc amplification was detected in 9 of the 15 A-RMSs but in none of the 14 E-RMSs. Tumor cells exhibiting N-myc amplification were identified only in the alveolar area in two A-RMSs, and they demonstrated a histologic mixture of alveolar and embryonal patterns. The remaining seven cases with an amplified N-myc showed a conventional alveolar pattern. Among the 15 A-RMSs, the survival rate of patients with tumors showing nonamplified N-myc and amplified N-myc oncogene was 4 (66%) of 6 and 1(11%) of 9, respectively (P < .05). No significant difference was observed between the other clinical findings (age, primary sites, clinical stages) of the N-myc-amplified and nonamplified tumors. Therefore, we concluded that the N-myc gene amplification, which is characteristic of a particular subtype of A-RMS, might be useful as a prognostic factor for an unfavorable outcome. PMID: 9872655 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 171: J Pediatr Surg. 1998 Nov;33(11):1695-8. Fluorescence in situ hybridization analysis of chromosome 1p36 deletions in human MYCN amplified neuroblastoma. Komuro H, Valentine MB, Rowe ST, Kidd VJ, Makino S, Brodeur GM, Cohn SL, Look AT. Department of Experimental Oncology, St Jude Children's Research Hospital, Memphis, Tennessee 38105-2794, USA. BACKGROUND/PURPOSE: Deletion of the short arm of chromosome 1 (1p) is one of the poor prognostic factors in human neuroblastomas. Recent studies have suggested that one or more of the neuroblastoma tumor suppressor genes reside in this region and have identified the shortest region of overlap (SRO) on 1p36. The purpose of this study was to examine deletions of 1p in human neuroblastomas by fluorescence in situ hybridization (FISH). METHODS: Two-color FISH analysis was performed to detect chromosome 1p36 abnormalities in 42 MYCN-amplified neuroblastomas. Four different probes from the 1p36 region, the E2F2, NPPA, D1S160, and CDC2L1 loci were used for detection of 1p abnormalities. A repeat sequence probe, which is specific for the heterochromatic region of chromosome 1 (pUC1.77), was used as a control. RESULTS: Large deletions of 1p36 were observed in 31 (73.8%) of 42 tumors, whereas the remaining 11 (26.2%) showed no deletion. In these 11 tumors, a translocation of 1p was found in one and a duplication of 1p was detected in another. CONCLUSIONS: A strong correlation between 1p abnormalities and MYCN amplification was found in this study. MYCN-amplified neuroblastomas were found to show large deletions of 1p encompassing the SRO. FISH provided a rapid and reliable method to detect hemizygous deletions of 1p. PMID: 9856898 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 172: Cancer Res. 1998 Dec 1;58(23):5396-405. Drug resistance patterns of human neuroblastoma cell lines derived from patients at different phases of therapy. Keshelava N, Seeger RC, Groshen S, Reynolds CP. Childrens Hospital Los Angeles, Department of Pediatrics, University of Southern California, 90033, USA. To determine whether neuroblastomas acquire a sustained drug-resistant phenotype from exposure to chemotherapeutic agents given to patients in vivo, we studied neuroblastoma cell lines established at different points of therapy: six at diagnosis before therapy (DX), six at progressive disease during induction therapy (PD-Ind), and five at relapse after intensive chemoradiotherapy and bone marrow transplantation (PD-BMT). Cells were maintained in the absence of drug selective pressure. Dose-response curves of melphalan, cisplatin, carboplatin, doxorubicin, and etoposide for the cell line panel were determined by measuring cytotoxicity with a 96-well-plate digital imaging microscopy (DIMSCAN) microassay. Drug resistance of cell lines progressively increased with the intensity of therapy delivered in vivo. The greatest resistance was seen in PD-BMT cell lines: IC90 values in PD-BMT cell lines were higher than clinically achievable drug levels by 1-37 times for melphalan, 1-9 times for carboplatin, 25-78 times for cisplatin, 6-719 times for doxorubicin, and 3-52 times for etoposide. Genomic amplification of MYCN did not correlate with resistance. Cross-resistance by Pearson correlation (r > or = 0.6) was observed between: (a) cisplatin + doxorubicin; (b) carboplatin + cisplatin, etoposide, or melphalan; (c) etoposide + cisplatin, melphalan, or doxorubicin. These data indicate that during therapy, neuroblastomas can acquire resistance to cytotoxic drugs because of the population expansion of tumor cells possessing stable genetic or epigenetic alterations that confer resistance. PMID: 9850071 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 173: Eur J Cancer. 1998 Aug;34(9):1391-7. 10 years' neuroblastoma screening in Europe: preliminary results of a clinical and biological review from the Study Group for Evaluation of Neuroblastoma Screening in Europe (SENSE). Erttmann R, Tafese T, Berthold F, Kerbl R, Mann J, Parker L, Schilling F, Ambros P, Christiansen H, Favrot M, Kabisch H, Hero B, Philip T. Department of Pediatric Hematology, Universitatskinderklinik, Hamburg, Germany. Between January 1986 and May 1996, 870,313 children were tested in European neuroblastoma (NB) screening programmes. Among these children, 82 cases of NB (age range 4-24 months, median 11 months) were detected by screening. 83% of the patients had localised NB and 17% were diagnosed with generalised NB (stage 4, 10%; stage 4s, 7%). Unfavourable biological markers (MYCN amplification, loss of heterozygosity (LOH) 1p36, DNA di/tetraploidy) were observed in 14% of 76 biologically examined cases. The median follow-up time of all the patients was 21.5 months (range 1-101 months). To date, 69 patients are in complete remission (CR) and 2 patients have died due to therapy (stage 4, 1 patient; stage 3, 1 patient with unfavourable markers). Apart from screened patients, 16 other patients with NB were found who had previously had a normal screening test, i.e. 'false negative' patients (age range 10-41 months, median 31.5 months). The median interval between screening and diagnosis was 24.5 months (range 6-35 months). 11 of the 'false negative' patients suffered from generalised NB (stage 4) and 5 had localised NB at diagnosis. Unfavourable biological markers were observed in 7/12 patients. 5 patients have died, 2 achieved partial remission and 9 CR. 9 of the 11 patients with unfavourable biological markers diagnosed due to NB screening are currently in CR. It is very likely that, among the patients without unfavourable biological markers, we detected tumours which may have regressed spontaneously. These children may have undergone 'unnecessary,' but unavoidable, diagnostic procedures and therapy. To reduce the number of 'false negative' patients, a later screening could be helpful and should be evaluated. PMID: 9849422 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 174: Genes Chromosomes Cancer. 1998 Dec;23(4):307-16. Mapping of chromosomal imbalances in gastric adenocarcinoma revealed amplified protooncogenes MYCN, MET, WNT2, and ERBB2. Nessling M, Solinas-Toldo S, Wilgenbus KK, Borchard F, Lichter P. Abteilung Organisation komplexer Genome, Deutsches Krebsforschungszentrum, Heidelberg, Germany. Gastric adenocarcinoma is a malignant tumor with a high incidence and a low survival rate. In order to identify genetic alterations associated with this tumor, we screened 23 gastric adenocarcinomas for recurrent chromosomal imbalances by using comparative genomic hybridization (CGH). The most common gains of chromosomal material were found on chromosome arms 20q (10 cases), 16p (7 cases), and 1q (4 cases) and on chromosome 11 (4 cases). Losses were observed on chromosome arms 4q, 5q, 9p, and 21q (3 cases each). Four tumors exhibited high-level amplifications localized on chromosome regions 2p23-p24, 7q31-q32, 8p21-p22, 10q25-q26, 11q13, 17q11-q21, and 20q. Based on the position of these amplifications, candidate (onco)genes were selected and subsequently tested by Southern blot analysis of the respective tumors. Of the seven tested candidates, MYCN, MET, WNT2, and ERBB2 were found to participate in the amplicons of the respective tumor samples. Of these four presumably activated oncogenes, two, MYCN and WNT2, were previously not assumed to play a pathogenic role in stomach cancer. Among the other regions of imbalance, gain of 20q seems particularly interesting, because it is found in almost half of the analyzed cases and is highly amplified. Our data allowed us to narrow the relevant region down to the commonly gained bands 20q12-q13.1. This and other imbalanced regions provide a basis for searching new putative oncogenes and tumor suppressor genes involved in the development or progression of gastric adenocarcinoma. PMID: 9824203 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 175: Clin Cancer Res. 1996 Aug;2(8):1361-7. Prognostic value of TrkA protein detection by monoclonal antibody 5C3 in neuroblastoma. Kramer K, Gerald W, LeSauteur L, Uri Saragovi H, Cheung NK. Departments of Pediatrics and Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. The effects of nerve growth factor, a neurotrophin mediating growth and differentiation of neural crest-derived cells, are mediated by the receptor TrkA. TrkA mRNA expression has been associated with a good prognosis in human neuroblastoma (NB). We describe the use of monoclonal antibody 5C3 in detecting TrkA expression by immunohistochemistry in NB and other malignant tumors. A murine anti-TrkA IgG1 monoclonal antibody, 5C3, was generated against the extracellular domain of human p140(TrkA). 5C3 detected a 140-kDa band on Western blots. 5C3 was optimized for immunostaining and used to detect p140(TrkA) on 113 frozen NB samples and 42 samples from nine other malignancies. MOPC21 IgG1 antibody was used as a control. Results by immunohistochemistry were compared to TrkA expression assessed by reverse transcription-PCR and Western analysis. The prognostic value of TrkA expression by these methods was evaluated and compared to other known prognostic variables, including stage, age, and MYCN copy number. TrkA expression was detected by immunohistochemistry in 73 of the 113 NB tumor specimens and strongly correlated with nonmetastatic disease. TrkA expression was specific for NB among small round blue cell tumors. Both TrkA expression by immunohistochemistry and localized/4s disease correlated with survival. Tumors from 55 of 60 patients with localized/4s NB exhibited homogeneous or a mixed pattern of TrkA immunohistochemistry, whereas only 18 of 53 patients with stage 4 NB were immunoreactive. Detection of TrkA by reverse transcription-PCR and Western analysis was much more sensitive and no longer correlated with survival. 5C3 enables rapid detection of p140(TrkA) by immuno-histochemistry and identifies patients more likely to have localized NB with a favorable clinical outcome. Lack of TrkA expression is correlated with metastatic, malignant NB. A subset of patients with NB, however, died of aggressive metastatic disease despite TrkA expression. As a mimic of nerve growth factor, 5C3 may be useful in the study of TrkA-expressing tumors. PMID: 9816308 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 176: Clin Cancer Res. 1997 Jul;3(7):1221-8. Clinical investigation of neuroblastoma with partial deletion in the short arm of chromosome 1. Ohtsu K, Hiyama E, Ichikawa T, Matsuura Y, Yokoyama T. First Department of Surgery and Department of General Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734, Japan. Several loci on the short arm of chromosome 1 (1p) have been reported as the consensus deleted regions for the putative suppressor genes of neuroblastoma by deletion mapping. The significance of deletion in 1p on the clinical features of neuroblastoma remains controversial. To clarify the relationship between the clinical features of neuroblastoma cases and genetic status of 1p, we performed deletion mapping on 1p on samples obtained from 58 cases with neuroblastoma using 12 highly polymorphic microsatellite or minisatellite loci. Loss of heterozygosity of 1p was detected in 19 cases (33%) of primary tumors and in 21 cases (36%) when metastatic and recurrent sites were included. They were classified into two groups according to the 1p deletion pattern: interstitial deletion (group I, n = 11) and terminal deletion (group T, n = 10). The shortest region of overlap in group I ranged between FGR and D1S170 (1p36.1-2). Clinically, all group I cases survived disease free, and none of these cases showed MYCN amplification. However, in group T, eight (80%) cases showed a large terminal deletion from D1S162 (1p32-pter), including the shortest region of overlap of group I, and two (20%) showed a very terminal deletion from D1S160 (1p 36.3). Of the group T cases, only two survived disease free, and seven (70%) showed MYCN amplification. Thus, the candidates for the locations of neuroblastoma suppressor genes on 1p may involve at least two regions, which demonstrate different clinical features. PMID: 9815803 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 177: Clin Cancer Res. 1997 Oct;3(10):1699-706. MYCN protein expression as a predictor of neuroblastoma prognosis. Chan HS, Gallie BL, DeBoer G, Haddad G, Ikegaki N, Dimitroulakos J, Yeger H, Ling V. Division of Hematology-Oncology and Immunology, Department of Pediatrics,The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, M5G 1X8 Canada. hlschan@resunix.ri.sickkids.on.ca About half of nonlocalized neuroblastomas have MYCN gene amplification and usually progress rapidly, but the half without such amplification also do poorly, albeit progressing more slowly. We hypothesize that overexpression of MYCN protein can occur without gene amplification and that this expression reliably predicts the prognosis of neuroblastoma. To determine whether MYCN expression correlated with outcome, we assayed MYCN protein immunohistochemically in 180 archival pretreatment and posttreatment samples and stratified the 57 conventionally treated stage IVS, III, and IV patients by these conventional prognostic factors: stage, age, serum ferritin, Shimada histology, urinary catecholamine ratio, and MYCN gene status. At a median follow-up of >/=6.8 years, we found in patients with known MYCN gene status that the 23 of 37 without gene amplification fared no better than the 14 of 37 with gene amplification (P = 0.35 and 0.21, comparing relapse-free and survival rates). Conversely, in patients without MYCN gene amplification, 9 of 23 were found to overexpress MYCN protein pretreatment, and they did worse than the 14 of 23 without detectable MYCN protein (P = 0.0016 and 0.022, comparing relapse-free and survival rates). Furthermore, MYCN protein expression was prognostic without (P = 0.00001) and with (P = 0.0007) stratifying all 57 patients by MYCN gene status, each conventional prognostic factor (P ranging from 0.00001-0.013), or simultaneously by the two most important factors, stage and age (P = 0.00076). We conclude that overexpression of MYCN protein without gene amplification correlated significantly with the clinical behavior of neuroblastoma and predicted outcome independently of other prognostic factors. This strongly supports the hypothesis that expression of the MYCN oncogene is critical for progression of neuroblastoma. PMID: 9815553 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 178: J Pediatr Hematol Oncol. 1998 Sep-Oct;20(5):489-93. Familial neuroblastoma: report of a kindred with later age at diagnosis. Lemire EG, Chodirker BN, Williams GJ, Seargeant LE, Israels SJ, Phillips SM, de Nanassy JA, Maris JM, Yanofsky RA. Department of Pediatrics, University of Manitoba, Winnipeg, Canada. PURPOSE: To describe the clinical and biologic features of neuroblastoma (NB) in two siblings and their maternal second cousin. PATIENTS AND METHODS: NB was diagnosed in the siblings at 2 1/2 (patient 2) and 5 (patient 3) years of age. NB was diagnosed in their maternal second cousin (patient 1) when she was 7 years old. Standard clinical and biological data, tumor karyotype, and tumor allelotype at select loci were obtained. RESULTS: Patient 1 had International Neuroblastoma Staging System (INSS) stage 4 NB and unfavorable histology but no evidence of MYCN amplification; she died from complications of autologous bone marrow transplantation in second remission. Patient 2 had INSS stage 4 NB with unfavorable histology but no MYCN amplification; her disease recurred 39 months after completing therapy. Patient 3 had INSS stage 1 NB with favorable biologic features; he was treated with surgical excision and remains free of disease. CONCLUSIONS: Familial NB may occur at a later age than predicted by the tumor suppressor gene model of inherited cancer. This report further emphasizes the clinical and biological heterogeneity of familial NB. Publication Types: Case Reports PMID: 9787327 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 179: J Pediatr Hematol Oncol. 1998 Sep-Oct;20(5):486-8. An infant with neuroblastoma and MYCN amplification found through mass screening. Taga T, Okamoto N, Hisano T, Tanaka T, Shimada M, Okabe H, Shimada H, Ohta S. Department of Pediatrics, Shiga University of Medical Science, Japan. PURPOSE: This report describes an extremely rare case of an infant with neuroblastoma involving MYCN amplification found through mass screening (MS). PATIENT: An 8-month-old boy was referred to our hospital after screening for neuroblastoma showed positive results. A firm tumor was found above his left kidney and completely resected. RESULTS: The tumor arose from the left adrenal gland. The disease was classified as stage I according to the International Neuroblastoma Staging System. Although the tumor showed a favorable histology, MYCN was amplified to 20 copies. After surgery, the infant underwent chemotherapy, but a new mass in the right adrenal gland was found. After chemotherapy, the tumor was excised. Despite several courses of chemotherapy, multiple metastases in both lung fields appeared. More intensive chemotherapy and allogenic peripheral blood stem cell transplantation were performed, but the infant died from pulmonary hemorrhage after the transplantation. CONCLUSIONS: The majority of patients with neuroblastoma found through MS have a good outcome. However, some patients have poor prognostic factors, like our patient with MYCN amplification. There is a need to develop a suitable protocol for babies with high risk features younger than 1 year of age found through MS. Publication Types: Case Reports PMID: 9787326 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 180: J Clin Oncol. 1998 Oct;16(10):3286-94. Prognostic significance of MYCN oncogene expression in childhood neuroblastoma. Bordow SB, Norris MD, Haber PS, Marshall GM, Haber M. Children's Cancer Research Institute, Sydney Children's Hospital, Randwick, NSW, Australia. PURPOSE: To assess the significance of MYCN gene expression as a prognostic factor in patients with neuroblastoma of various ages, and to determine whether it can predict for outcome independently of MYCN gene amplification. PATIENTS AND METHODS: The level of MYCN gene expression in 60 specimens of primary untreated neuroblastoma was determined by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: High levels of MYCN gene expression were associated with advanced tumor stage (P=.0005), with the presence of MYCN gene amplification (P < .0001), but not with older age at diagnosis. Among patients who lacked MYCN gene amplification, the levels of MYCN gene expression were significantly greater in the tumors of infants compared with those of older children (P < .0005). High MYCN expression was strongly associated with reduced survival and event-free survival in the overall study population (P < .005), and also in the subset of patients aged older than 1 year at diagnosis (P < .001). In contrast, MYCN expression did not appear to be predictive of outcome in infants. After adjustment for the effect of MYCN amplification, high levels of MYCN expression retained significant prognostic value for poor survival (relative hazards, 30.3; P=.003) in children aged older than 12 months at diagnosis. CONCLUSION: High MYCN gene expression is strongly predictive of poor outcome in older children with neuroblastoma, but not in infants. The findings help explain the controversy in the literature about the prognostic value of MYCN gene expression and highlight the different biology of neuroblastoma that presents in infants and older children. Publication Types: Review PMID: 9779703 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 181: Bull Cancer. 1998 Sep;85(9):739. Fiche n 7 : MYCN (N-myc) [No authors listed] PMID: 0009770592 [PubMed - as supplied by publisher] --------------------------------------------------------------- 182: Cancer Lett. 1998 Aug 14;130(1-2):83-92. Interstitial and large chromosome 1p deletion occurs in localized and disseminated neuroblastomas and predicts an unfavourable outcome. Iolascon A, Lo Cunsolo C, Giordani L, Cusano R, Mazzocco K, Boumgartner M, Ghisellini P, Faienza MF, Boni L, De Bernardi B, Conte M, Romeo G, Tonini GP. Department of Biomedicine of Evolutive Age, University of Bari, Genova, Italy. We studied chromosome 1p loss of heterozygosity (1p-LOH) in 53 neuroblastomas (NBs) using 15 (CA)n repeat loci, which covered a region of 90 cM. We also assessed chromosome 1p36 deletion by fluorescence in situ hybridization (FISH) on interphase nuclei. 1p-LOH was found in 19 (36%, 95% confidence interval (CI) 23-50%) NBs. We detected interstitial and large deletion in both localized and disseminated tumours and in one tumour of a patient at stage 4S. Allelic loss was frequently observed in 1p36 and 1p32 regions. In patients older than 1 year of age (53 versus 13%, P < 0.002) we detected significant chromosome 1p deletion and it was associated with MYCN amplification (P = 0.001). Overall survival (OS) analysis showed that 1p-LOH is predictive of a poor outcome (odds ratio 16.5, 95% CI 5.4-50.9%); therefore, 1p-LOH should be regarded as an additional tumour progression marker in neuroblastoma. PMID: 9751260 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 183: Genes Chromosomes Cancer. 1998 Oct;23(2):134-40. MYCN is the only highly expressed gene from the core amplified domain in human neuroblastomas. Reiter JL, Brodeur GM. Division of Oncology, The Children's Hospital of Philadelphia and the University of Pennsylvania 19104-4318, USA. MYCN amplification in neuroblastomas is strongly associated with advanced stages of disease and a poor prognosis. We have recently defined a 130 kb core region of the MYCN amplicon that is consistently amplified in neuroblastomas. However, it has been argued that other expressed sequences were coamplified with MYCN and, as a result, might contribute to the aggressive phenotype of MYCN-amplified neuroblastomas. Therefore, we have screened cosmids representing the core MYCN-amplified domain and surrounding DNA by using a differential hybridization approach to detect other amplified, highly expressed genes from this region. Our results suggest that MYCN is the only highly expressed gene consistently amplified in human neuroblastomas, and that the MYCN gene is likely to be the only selective marker for genomic amplification in these tumors. PMID: 9739016 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 184: Oncogene. 1998 Jul 23;17(3):339-46. MycN and IFNgamma cooperate in apoptosis of human neuroblastoma cells. Lutz W, Fulda S, Jeremias I, Debatin KM, Schwab M. Department of Cytogenetics-0825, German Cancer Research Center, Heidelberg. Neuroblastomas undergo spontaneous regression at an unusually high rate. The mechanisms are not clear, but apoptosis may be involved. A large proportion of neuroblastomas is characterized by amplification of MYCN. Using human neuroblastoma cells harbouring tetracycline controlled expression of MYCN we have analysed the role of the MycN protein and IFNgamma in cell death decision. Neither conditional expression of MYCN nor treatment with IFNgamma alone was sufficient to trigger cell death. However, when acting in concert MycN and IFNgamma efficiently triggered cell death, which was accompanied by DNA fragmentation and required caspase activity, two hallmarks of apoptosis. MycN and IFNgamma may cooperate along at least two different pathways. First, IFNgamma increased the CD95 cell surface expression while MycN enhanced the cellular susceptibility for the CD95 mediated death signal. Second, IFNgamma treatment induced expression of BAK mRNA while MycN and IFNgamma in combination increased the amount of Bax protein, another activator of apoptosis, without a concomitant increase in BAX mRNA. MycN also increased cell death in response to TRAIL and TNFalpha, suggesting that enforced MYCN expression in general increases the susceptibility of neuroblastoma cells towards a variety of death stimuli. PMID: 9690515 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 185: Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9172-7. Identification of a large Myc-binding protein that contains RCC1-like repeats. Guo Q, Xie J, Dang CV, Liu ET, Bishop JM. G. W. Hooper Foundation, University of California, San Francisco, CA 94143, USA. guoq@pop.nci.nih.gov The protooncogene MYC plays an important role in the regulation of cellular proliferation, differentiation, and apoptosis and has been implicated in a variety of human tumors. MYC and the closely related MYCN encode highly conserved nuclear phosphoproteins (Myc and NMyc) that apparently function as transcription factors in the cell. We have identified a large and highly conserved nuclear protein that interacts directly with the transcriptional activating domain of Myc (designated "protein associated with Myc" or Pam). Pam contains an extended amino acid sequence with similarities to a protein known as regulator of chromosome condensation (RCC1), which may play a role in the function of chromatin. The gene encoding Pam (PAM) is expressed in all of the human tissue examined, but expression is exceptionally abundant in brain and thymus. Pam binds specifically to Myc, but not NMyc. The region in Myc required for binding to Pam includes a domain that is essential for the function of Myc and that is frequently mutated in Burkitt's lymphomas. PAM is located within a 300-kb region on chromosome 13q22. PMID: 9689053 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 186: Anticancer Res. 1998 May-Jun;18(3A):1793-7. In vitro effects of MYCN sense and antisense expression in MYCN-amplified human neuroblastoma cells. Kavallaris M, Gardaneh M, Cheung B, Camacho ML, Hocker JE, Norris MD, Haber M, Marshall GM. Children's Cancer Research Institute, Sydney Children's Hospital, Randwick, Australia. Amplification of the MYCN oncogene is a strong predictor of treatment failure and chemo-resistance in childhood neuroblastoma. Stable expression of two partial MYCN gene fragments in antisense orientation reduced Mycn protein expression in an MYCN-amplified neuroblastoma tumor cell line, however, antisense cells did not exhibit an increased in vitro sensitivity to cytotoxic or differentiating agents. In contrast, partial MYCN sense transfectants exhibited increased resistance to cytotoxic drugs. These data suggest that the chemo-resistance of MYCN-amplified neuroblastoma cells is complex, and may be due to factors additional to Mycn protein expression. PMID: 9673406 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 187: Br J Cancer. 1998 Jun;77(11):1787-91. Loss of heterozygosity of 3p markers in neuroblastoma tumours implicate a tumour-suppressor locus distal to the FHIT gene. Ejeskar K, Aburatani H, Abrahamsson J, Kogner P, Martinsson T. Department of Clinical Genetics, Gothenburg University, Sahlgrenska University Hospital/Ostra, Sweden. Neuroblastoma is a heterogeneous childhood tumour of the sympathetic nervous system, in which deletions of chromosomal region 1p and amplification of the MYCN oncogene correlate with aggressive tumour behaviour. However, the majority of neuroblastoma tumours show neither of these aberrations, indicating that other chromosomal regions may be involved in tumorigenesis. Here, we report findings of loss of heterozygosity (LOH) on chromosome 3. In our neuroblastoma material, nine of 59 (15.3%) tested tumours showed allelic loss of chromosome 3p markers. We found significant clinical and biological differences between tumours with the loss of one entire chromosome 3 vs tumours with partial loss in chromosome region 3p. All children with tumours with whole chromosome 3 loss are long-term survivors, whereas all children with tumours showing partial 3p LOH have died from tumour progression. A consensus region found to be deleted in all the tumours with 3p deletions was defined by markers D3S1286 and D3S1295, i.e. 3p25.3-p14.3, distal to the FHIT gene. PMID: 9667647 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 188: J Clin Oncol. 1998 Jun;16(6):2007-17. Prognostic significance of age, MYCN oncogene amplification, tumor cell ploidy, and histology in 110 infants with stage D(S) neuroblastoma: the pediatric oncology group experience--a pediatric oncology group study. Katzenstein HM, Bowman LC, Brodeur GM, Thorner PS, Joshi VV, Smith EI, Look AT, Rowe ST, Nash MB, Holbrook T, Alvarado C, Rao PV, Castleberry RP, Cohn SL. Department of Pediatrics, Northwestern University, Chicago, IL, USA. PURPOSE: Although a high rate of spontaneous regression is observed in infants with stage D(S) neuroblastoma (NB), survival is not uniform. To determine the prognostic relevance of age at diagnosis, therapy, and tumor biology in infants with stage D(S) NB, we reviewed the Pediatric Oncology Group (POG) experience. PATIENTS AND METHODS: A review of patients diagnosed with stage D(S) NB registered on POG protocols was performed. Survival according to age at diagnosis, treatment, and tumor biology was determined. RESULTS: Between 1987 and 1996, 110 infants with stage D(S) NB had an estimated 3-year survival rate of 85% +/- 4%; survival rate was 71% +/- 8% for infants 2 months of age or younger, and 68% +/- 12%, 44% +/- 33%, and 33% +/- 19% for patients with diploid, MYCN-amplified, and unfavorable histology tumors, respectively. Survival rates were similar for patients who received adjuvant chemotherapy versus those who did not (82% +/- 5% v 93% +/- 6%, respectively; P = .187). Furthermore, there was no statistical difference in survival rate for patients who underwent complete resection of their primary tumor compared with those who underwent partial resection or biopsy only (90% +/- 5% v 78% +/- 7%, respectively; P = .083). CONCLUSION: Our review confirmed that the survival of infants with stage D(S) NB is excellent. However, subsets of patients with poor prognosis can be identified by young age and unfavorable biologic factors. More effective therapy is needed for the group of stage D(S) infants who show unfavorable clinical and biologic features. PMID: 9626197 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 189: Anticancer Res. 1998 Mar-Apr;18(2A):1211-5. Differential polymerase chain reaction with serial dilutions for quantification of MYCN gene amplification in neuroblastoma. Gallego S, Reventos J, Sanchez de Toledo J, Munell F. Centre d'Investigacions en Bioquimica i Biologia Molecular, Hospital Universitari Vall d'Hebron, Barcelona, Spain. sgallego@ar.vhebron.es BACKGROUND: MYCN amplification is a powerful prognostic marker in neuroblastoma. Since MYCN status guides therapy results should be available promptly after diagnosis. MATERIAL AND METHODS: We used a differential PCR assay to analyze neuroblastoma samples obtained from 25 patients diagnosed and treated at our Institution. Serial dilutions of test DNA in control DNA were performed prior to differential PCR in order to quantitate the MYCN copy number. RESULTS: MYCN amplification was identified by differential PCR in five samples out of twenty five. The serial dilutions of amplified DNAs performed before the PCR reaction allowed a precise estimation of the copy number in the 5 samples with amplification. CONCLUSIONS: The present results confirm differential PCR assay as an easy, sensitive and rapid technique to evaluate the MYCN gene amplification in neuroblastoma. Serial dilutions accurately estimate the gene copy number allowing the early onset of the appropriate treatment. PMID: 9615790 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 190: Eur J Cancer. 1997 Oct;33(12):2058-63. Coexpression of mRNA for the full-length neurotrophin receptor trk-C and trk-A in favourable neuroblastoma. Svensson T, Ryden M, Schilling FH, Dominici C, Sehgal R, Ibanez CF, Kogner P. Department of Woman and Child Health, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden. Neuroblastoma, a childhood tumour of the sympathetic nervous system, may sometimes regress spontaneously in infants, or progress to a poor clinical outcome despite intensive therapy. Neuroblastomas express neurotrophin receptors and high levels of mRNA for trk-A correlates with favourable outcome, whereas trk-B mRNA is expressed by more unfavourable tumours. Using a sensitive RNase protection assay, mRNA expression for the neurotrophin receptor trk-C was investigated in 50 tumour samples from 45 children at different stages including metastatic and relapsing tumour tissue, out of which 22 were also investigated for trk-A mRNA. Thirty-seven of 43 primary tumours (86%) showed trk-C mRNA with more than 300-fold difference between the highest and the lowest values. A higher trk-C index (trk-C mRNA/GAPDH mRNA) was associated with favourable features such as younger age (P = 0.009-0.003), favourable tumour stage (1, 2 or 4S; P < 0.001) and favourable prognosis (P = 0.044). Better survival probability was shown in children with intermediate or high trk-C index compared with patients with low or undetectable levels (P = 0.031). All localised tumours co-expressed mRNA for trk-A and trk-C receptors. RT-PCR analysis detected mRNA encoding the cytoplasmic trk-C tyrosine kinase region only in favourable neuroblastomas. We conclude that favourable neuroblastoma may express the full-length trk-C receptor while unfavourable tumours, especially those with MYCN amplification, seem to either express no trk-C or truncated trk-C receptors with unknown biological function. Trk-C and possibly its preferred ligand NT-3 may be involved in the biology of favourable neuroblastomas showing apoptosis or differentiation. PMID: 9580079 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 191: Eur J Cancer. 1997 Oct;33(12):2101-5. Clinical relevance of CD44 cell surface expression and MYCN gene amplification in neuroblastoma. Combaret V, Gross N, Lasset C, Frappaz D, Beretta-Brognara C, Philip T, Beck D, Favrot MC. Department of Tumour Biology, Centre Leon Berard, Lyon, France. This multicentric analysis of tumours obtained from 140 patients with neuroblastoma confirms that the lack of CD44 expression is a highly significant factor of poor prognosis and, as previously published in multivariate analysis of the four factors, i.e. MYCN amplification, CD44 expression, age and tumour stage, CD44 expression and tumour stage were the only independent prognostic factors of event-free survival (Combaret et al., J Clin Oncol 1996, 14, 25-34). Furthermore, CD44 analysis affords significant prognostic discrimination in subgroups of patients with or without MYCN amplified tumours, both in low-stage neuroblastomas and high-grade neuroblastomas. In the subgroup of patients with low-stage neuroblastoma and the stage 4 subgroup, CD44 was the only independent prognostic factor for the prediction of event-free survival in a multivariate analysis. In conclusion, CD44 is one of the most powerful factors for predicting clinical outcome in neuroblastoma at the time of initial staging. Publication Types: Multicenter Study PMID: 9516862 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 192: Eur J Cancer. 1997 Oct;33(12):2098-100. Correlation of MYCN amplification, Trk-A and CD44 expression with clinical stage in 250 patients with neuroblastoma. Kramer K, Cheung NK, Gerald WL, LaQuaglia M, Kushner BH, LeClerc JM, LeSauter L, Saragovi HU. Department of Pediatrics and Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. In contrast to MYCN amplification, expression of either trk-A or CD44 in neuroblastoma is a favourable prognostic factor and were therefore investigated in tumours from 250 patients. One hundred and eleven localised/4s (Group 1) and 139 stage 4 (Group 2) tumours were analysed. MYCN copy number was obtained by Southern blotting or PCR amplification and was detected in 28 stage 4 tumours. Trk-A and CD44 expression was detected by immunoperoxidase staining using murine monoclonal antibodies 5C3 and L178, respectively. Expression was scored as positive (homogeneous), mixed (heterogeneous) or non-reactive (negative). Trk-A expression was found in 95% of Group 1 tumours and 49% of Group 2 tumours. CD44 expression was found in 100% of Group 1 tumours, the majority of which had a strong homogeneous expression. Lack of CD44 expression occurred in 25% of tumours, all stage 4 neuroblastoma. Of the 28 MYCN amplified tumours, 27/28 (96%) were trk-A negative, and 13/28 (46%) CD44 negative. We conclude that (1) a significant percentage of stage 4 neuroblastoma express either or both trk-A and CD44, (2) the absence of CD44 expression is highly restricted to stage 4 neuroblastoma and is associated with the lack of trk-A expression, (3) trk-A negativity among CD44-positive tumours is associated with stage 4 neuroblastoma and (4) the absence of trk-A expression is highly correlated with MYCN amplification. PMID: 9516861 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 193: Eur J Cancer. 1997 Oct;33(12):2092-7. The current contribution of molecular factors to risk estimation in neuroblastoma patients. Berthold F, Sahin K, Hero B, Christiansen H, Gehring M, Harms D, Horz S, Lampert F, Schwab M, Terpe J. Klinik und Poliklinik fur Kinderheilkunde der Universitat zu Koln, Germany. The association of molecular characteristics with prognosis has been reported, but not their relationship with each other and their impact in the context of known clinical risk factors. In this study, data of 1249 consecutive intent-to-treat-neuroblastoma patients with more than 1 year follow-up were examined by multivariate analysis using loglinear and Cox proportional hazard regression models on a stage-related basis (stages 1-3: 600, 4S: 116, 4: 533). In a first step, risk factors were identified from 18 selected clinical variables, and risk groups defined. The second step investigated whether molecular characteristics (MYCN, LOH 1p, del 1p, CD44, N-ras, NGF-R, bcl-2, APO-1 (CD95)) contributed additional prognostic information to the model. The loglinear model demonstrated several interactions between clinical factors. By the Cox regression model, seven independent clinical risk factors were found for stages 1-3, seven for stage 4 and two for stage 4S. By subsequent introduction of all molecular variables, MYCN amplification only added significant prognostic information to the clinical factors in localised and stage 4 neuroblastoma. The models allowed the definition of risk groups for stages 1-3 patients by age (e beta = 5.09) and MYCN (e beta = 4.26), for stage 4 by MYCN (e beta = 2.78) and number of symptoms (e beta = 2.44) and for stage 4S by platelet count (e beta = 3.91) and general condition (e beta = 2.99). Molecular factors and in particular MYCN contribute significantly to risk estimation. In conjunction with clinical factors, they are powerful tools to define risk groups in neuroblastoma. PMID: 9516860 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 194: Eur J Cancer. 1997 Oct;33(12):2084-9. Somatostatin in neuroblastoma and ganglioneuroma. Kogner P, Borgstrom P, Bjellerup P, Schilling FH, Refai E, Jonsson C, Dominici C, Wassberg E, Bihl H, Jacobsson H, Theodorsson E, Hassan M. Dept. of Woman and Child Health, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden. Neuroblastoma, a childhood tumour of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. Somatostatin may inhibit neuroblastoma growth and induce apoptosis in vitro and was therefore investigated. Using a radioimmunoassay, we found that all ganglioneuromas contained high somatostatin concentrations (> 16 pmol/g), significantly higher than neuroblastomas (n = 117, median 2.8 pmol/g), healthy adrenals, Wilms' tumours, phaeochromocytomas and other neuroendocrine tumours (P < 0.001). Neuroblastomas contained more somatostatin than control tumours (P < 0.001-0.05). Neuroblastomas amplified for the MYCN oncogene contained less somatostatin than non-amplified tumours (1.2 pmol/g versus 4.0 pmol/g, respectively; P = 0.026). In a clinically unfavourable neuroblastoma subset (age > 12 months, stage 3 or 4) 16 children with high concentrations of somatostatin in primary tumours had a better prognosis than 23 with low somatostatin (46.7% versus 0% survival at 5 years, P < 0.005). Scintigraphy using 111In-pentetreotide identified tumours expressing high-affinity somatostatin receptors in vivo. However, no significant correlation was found between somatostatin receptor expression and peptide content in 15 tumours. Similarly, human SH-SY5Y neuroblastoma xenografts grown in nude rats showed low somatostatin concentrations, but were positive for somatostatin receptor scintigraphy. Treatment of these rats with the somatostatin analogue octreotide seemed to upregulate in vivo receptor expression of somatostatin and vasoactive intestinal peptide more effectively than 13-cis retinoic acid. In conclusion, somatostatin in neuroblastoma is associated with differentiation to benign ganglioneuromas in vivo and favourable outcome in advanced tumours. Furthermore, somatostatin receptor scintigraphy may identify tumours with high-affinity receptors in children that might benefit from targeted therapy using synthetic somatostatin analogues. PMID: 9516858 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 195: Eur J Cancer. 1997 Oct;33(12):2081-3. Interleukin-1 beta converting enzyme (ICE) is preferentially expressed in neuroblastomas with favourable prognosis. Ikeda H, Nakamura Y, Hiwasa T, Sakiyama S, Kuida K, Su MS, Nakagawara A. Department of Surgery, Gunma Children's Medical Centre, Japan. To determine whether interleukin-1 beta converting enzyme (ICE) plays a role in the programmed cell death of neuroblastoma, we studied ICE expression in primary tumours. In patients in stages I, II and IVS, ICE mRNA was detected in 22 of 32 (69%) tumours, while only 5 of 26 (19%) tumours expressed ICE in stages III and IV (P < 0.001). ICE mRNA was expressed in 27 of 47 (57%) tumours without MYCN amplification, but it was not detected in any tumours with MYCN amplification (P < 0.01). Immunohistochemically, the cytoplasm was stained in all 15 neuroblastomas examined. The nuclei were stained in 12 neuroblastomas without MYCN amplification, whereas only 1 of 3 tumours with MYCN amplification had positive staining in the nuclei. In ganglioneuromas, high levels of ICE mRNA were expressed, but immunostaining showed that the protease expression was confined to the cytoplasm. These observations suggest that ICE may be associated with the spontaneous regression often seen in favourable neuroblastomas and that localisation of ICE protease in the cell may be important for the cell death pathway. Double staining for ICE and TUNEL showed that they were co-localised in some nuclei, but the distribution of ICE protease expression was not necessarily the same as that of DNA fragmentation, suggesting that the protease expression probably preceded DNA fragmentation during the apoptotic process. ICE may play an important role in regulating the apoptotic process of neuroblastoma. PMID: 9516857 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 196: Eur J Cancer. 1997 Oct;33(12):2071-4. HuD, a neuronal-specific RNA-binding protein, is a potential regulator of MYCN expression in human neuroblastoma cells. Ross RA, Lazarova DL, Manley GT, Smitt PS, Spengler BA, Posner JB, Biedler JL. Department of Biological Sciences, Fordham University, Bronx, New York 10458, USA. HuD is one of a family of neural antigens recognised by the sera of patients with antibody-associated paraneoplastic encephalomyelitis. Localised exclusively to neurons, these proteins are among the earliest markers of the developing nervous system. Sequence analysis suggests that HuD is an RNA-binding protein. Hu protein levels were determined for the three cell types characterising human neuroblastoma cell lines: sympathoadrenal neuroblasts (N), substrate-adherent Schwann/glial/melanoblastic precursors (S) and stem cells (I) which can give rise to both N and S cells. Western blot analysis showed similar levels of protein in three N-type cell lines; S cells have no detectable Hu protein. Northern blot analysis indicated that N cells express all three Hu genes, HuD, HuC and Hel-N1. N cells, mostly from MYCN-amplified cell lines, have consistently higher steady-state levels of MYCN mRNA than S cell counterparts. Nuclear run-on and mRNA half-life experiments revealed no differences in transcription rate or mRNA stability between N and S cells from the LA-N-1 cell line, implicating differences in post-transcriptional regulation. HuD is postulated to be instrumental in splicing/processing and/or stabilisation of mRNAs involved in cell growth and neuronal differentiation. As determined by gel-mobility shift assays, HuD fusion protein binds to the 3'UTR of human MYCN mRNA. Analysis of HuD deletion mutants has demonstrated that the first and second RNA-recognition motifs (RRMs) are required for binding. Whether HuD regulates MYCN expression and thereby influences tumour aggressiveness is of major interest. PMID: 9516855 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 197: Eur J Cancer. 1997 Oct;33(12):2064-7. Activity of a 40 kDa RNA-binding protein correlates with MYCN and c-fos mRNA stability in human neuroblastoma. Chagnovich D, Cohn SL. Program in Tumor Cell Biology, Robert H. Lurie Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611, USA. Subclones of neuroblastic (N) and substrate adherent (S) cells have been established from neuroblastoma tumours cultured in vitro which differ in growth characteristics and MYCN expression. N cells derived from the NBL-W cell line (W-N) express 5-fold higher levels of MYCN mRNA and 10-fold higher levels of MYCN protein than S cells (W-S), despite having the same MYCN copy number. In an effort to identify the molecular mechanisms responsible for the disparity in steady-state MYCN levels, the rate of MYCN mRNA degradation was measured in the two subclones. The half-life of MYCN mRNA in the W-N cells was approximately 45 min compared to approximately 6 min in the W-S cells. Similarly, the half-life of another labile mRNA, c-fos, differed in W-N and W-S cells (30 min versus 15 min, respectively). The turnover of labile mRNAs is thought to be mediated by the interactions of trans-acting factors with AU-rich elements within the 3' untranslated region. RNA UV cross-linking assays using W-N cell lysate demonstrated abundant quantities of a protein, 40 kDa in size (p40), that bound specifically to AU-rich elements within the MYCN and c-fos 3' untranslated region. However, p40 was barely detectable in W-S cells. Our studies suggest that p40 may play a role in determining neuroblastoma phenotype by regulating MYCN and c-fos mRNA turnover. PMID: 9516853 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 198: Eur J Cancer. 1997 Oct;33(12):2054-7. Expression and function of Trk-C in favourable human neuroblastomas. Yamashiro DJ, Liu XG, Lee CP, Nakagawara A, Ikegaki N, McGregor LM, Baylin SB, Brodeur GM. Division of Oncology, Children's Hospital of Philadelphia, Pennsylvania 19104-4318, USA. Human neuroblastomas express the neurotrophin receptors trk-A and trk-B. Favourable outcome is associated with expression of trk-A, while unfavourable, MYCN amplified tumours express trk-B. In this study we examined the expression of trk-C in primary neuroblastoma tumour-derived cell lines. We found by Northern blot analysis that trk-C mRNA is expressed in 14 of 55 (25%) primary tumours. Trk-C was expressed in significantly more lower stage tumours (stage 1, 2, 4S) than higher stage tumours (stage 3, 4, P < 0.04). The expression of trk-C was correlated positively with survival and negatively correlated with MYCN amplification. We also studied the function of trk-C in transfected cell lines and found that NT-3 promotes both cell survival and differentiation. Our results suggest that trk-C is involved in the biology of favourable neuroblastomas. PMID: 9516852 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 199: Eur J Cancer. 1997 Oct;33(12):2043-9. Neuroblastoma cells can actively eliminate supernumerary MYCN gene copies by micronucleus formation--sign of tumour cell revertance? Ambros IM, Rumpler S, Luegmayr A, Hattinger CM, Strehl S, Kovar H, Gadner H, Ambros PF. Children's Cancer Research Institute CCRI, St. Anna Kinderspital, Vienna, Austria. Human neuroblastoma cell lines frequently exhibit MYCN amplification and many are characterised by the presence of morphologically distinct cell types. The neuronal cells (N-cells) and the so-called flat cells (F-cells) are thought to represent manifestations of different neural crest cell lineages and are considered to be the consequence of neuroblastoma cell pluripotency. In this study, various neuroblastoma cell lines were examined for micronuclei. In F-cells of neuroblastoma cell lines with extrachromosomally amplified MYCN, we observed the frequent occurrence of micronuclei. Using fluorescence in situ hybridisation (FISH) with a MYCN specific probe, we demonstrated that these micronuclei were packed with MYCN hybridisation signals. In addition, in a minor percentage of cells, MYCN signals occurred in clusters, adhered to the nuclear membrane and aggregated in nuclear protrusions. In F-cells, a substantial reduction or lack of amplified MYCN copies was observed. These observations let us conclude that extrachromosomally amplified genes can be actively eliminated from the nucleus resulting in a dramatic loss of amplified sequences in the F-cells. Moreover, reduction or loss of amplified sequences in F-cells was shown to be accompanied by downregulation of MYCN expression, by a decrease in proliferative activity and by upregulation of molecules of the major histocompatibility complex class I (MHC I). Interestingly, F-cells are not restricted to neuroblastoma cell cultures, but also occur in cell lines of other tissue origin. All F-cells share important biological features, interpreted as cell revertance, i.e. loss of the malignant phenotype and properties. This fact, together with the demonstration that neuroblastoma cells do not differentiate into Schwann cells in vivo [1] Ambros et al. NEJM 1996, 334, 1505-1511, do not support the hypothesis that F-cells represent Schwannian/glial differentiation in vitro. We therefore postulate that the elimination of amplified MYCN gene copies in cultivated neuroblastoma cells is in line with the phenomenon of tumour cell revertance. PMID: 9516850 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 200: Eur J Cancer. 1997 Oct;33(12):2037-42. Analysis of candidate gene co-amplification with MYCN in neuroblastoma. George RE, Kenyon R, McGuckin AG, Kohl N, Kogner P, Christiansen H, Pearson AD, Lunec J. Cancer Research Unit, Newcastle University Medical School, University of Newcastle upon Tyne, U.K. Previous studies have revealed that the MYCN gene spans approximately 7kb, while the amplicon has been estimated to be 100 kb to 1 Mb long [1-3]. This implies that several other genes may be present on the MYCN amplicon. Such co-amplified genes could contribute to the malignant phenotype and might provide an explanation for why not all patients with MYCN amplification have a poor outcome. We investigated 7 neuroblastoma cell lines and 167 primary tumours for the co-amplification of candidate genes known to be present near the MYCN locus: ornithine decarboxylase, ribonucleotide reductase, syndecan-1 and a DEAD box protein gene, DDX1. We also investigated further the pG21 expressed sequence previously reported to be co-amplified with MYCN. No co-amplification with the first 3 genes was found in any of the cell lines or tumour samples. DDX1 was found to be amplified along with MYCN in 4/6 (67%) cell lines and 18/38 (47%) of the MYCN amplified tumours. No amplification of DDX1, ODC1, RRM2 or syndecan-1 was found in the absence of MYCN amplification. DDX1 co-amplification was observed to occur mainly in stage 4 or 4S patients. With the exclusion of those with 4S disease, patients with DDX1 co-amplification had a significantly shorter mean (+/- SE) disease-free interval (4.1 +/- 1.4, n = 8 months) compared with patients with MYCN amplification alone (19.6 +/- 4.5, n = 17) (P = 0.04, Welch's unpaired t-test). The pG21 sequence was identical to part of the DDX1 gene. These observations indicate that there is at least 1 other gene co-amplified with MYCN in a proportion of cases and that those patients with DDX1 co-amplification tend to relapse more quickly. It also implies that the MYCN amplicon is of varied size and/or position relative to the MYCN gene. PMID: 9516849 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 201: Eur J Cancer. 1997 Oct;33(12):2031-6. The prognostic value of MDR1 gene expression in primary untreated neuroblastoma. Haber M, Bordow SB, Haber PS, Marshall GM, Stewart BW, Norris MD. Children's Cancer Research Institute, Sydney Children's Hospital, Randwick, N.S.W., Australia. The contribution of MDR1 gene expression to the biology of childhood neuroblastoma is unclear. To clarify the role of MDR1 in this malignancy, we examined the relationship between MDR1 expression and patient outcome in subsets of 60 primary untreated neuroblastomas for which MYCN gene copy number and expression of the multidrug resistance-associated-protein (MRP) gene had been previously characterised. In contrast to MRP gene expression, MDR1 expression was lower in tumours with MYCN gene amplification compared with those without amplification. Strong correlations between MDR1 and MRP gene expression, and between MDR1 and MYCN gene expression, were observed in tumours lacking MYCN gene amplification (P < 0.0005). In these single-copy tumours, very high MDR1 gene expression was significantly associated with poor outcome (P < 0.05). Very high MDR1 expression was also strongly predictive of poor outcome in older children (P < 0.0001), but not in infants. These findings suggest a clinical role for the MDR1 gene in specific subgroups of primary neuroblastoma. PMID: 9516848 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 202: Eur J Cancer. 1997 Oct;33(12):1979-82. Sensitive and reliable detection of genomic imbalances in human neuroblastomas using comparative genomic hybridisation analysis. Van Gele M, Van Roy N, Jauch A, Laureys G, Benoit Y, Schelfhout V, De Potter CR, Brock P, Uyttebroeck A, Sciot R, Schuuring E, Versteeg R, Speleman F. Department of Medical Genetics, University Hospital, Ghent, Belgium. Deletions of the short arm of chromosome 1, extra copies of chromosome 17q and MYCN amplification are the most frequently encountered genetic changes in neuroblastomas. Standard techniques for detection of one or more of these genetic changes are karyotyping, FISH analysis and LOH analysis by Southern blot or PCR. Each of these techniques has its own particular limitations. More recently, comparative genomic hybridisation (CGH) was introduced for detection of genomic imbalances including deletions, duplications and gene amplification. We evaluated the sensitivity and reliability of CGH for detection of the most frequently encountered genetic changes in neuroblastoma. For this purpose a panel of well-characterised neuroblastoma cell lines as well as a series of 11 primary neuroblastomas was analysed. Our results show that CGH is a valuable tool for the genetic characterisation of neuroblastomas, both for the detection of frequently occurring genomic imbalances and for the identification of previously unnoticed genetic changes. PMID: 9516837 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 203: Eur J Cancer. 1997 Oct;33(12):1953-6. Loss of heterozygosity for chromosome 1p in familial neuroblastoma. Tonini GP, Lo Cunsolo C, Cusano R, Iolascon A, Dagnino M, Conte M, Milanaccio C, De Bernardi B, Mazzocco K, Scaruffi P. Unit of Solid Tumour Biology, G. Gaslini Institute/Advanced Biotechnology Centre, Genoa, Italy. Loss of heterozygosity (LOH) and deletion of chromosome 1p are very often found in sporadic neuroblastoma. Nevertheless, very few data are available concerning 1p LOH in familial neuroblastoma. Families with recurrent neuroblastoma are rare and analysis of chromosome 1p in these families might give useful information for identifying the putative neuroblastoma suppressor gene. We used combined cytogenetic and molecular techniques to study 1p LOH in two neuroblastoma families. Family M has 2 out of 3 children with neuroblastoma and family C has 2 children, 1 of whom has neuroblastoma and type 1 neurofibromatosis (NF1). All patients of both families showed tumour cells with chromosome 1p deletion (1pdel), but only the patient from family C also had MYCN gene amplification. In all cases the deleted chromosome 1 was of maternal origin. Publication Types: Case Reports PMID: 9516831 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 204: Eur J Cancer. 1997 Oct;33(12):1949-52. Restriction fragment length polymorphism analysis reveals different allele frequency and a linkage disequilibrium at locus D1S94 in neuroblastoma patients. Perri P, Pession A, Mazzocco K, Scaruffi P, Strigini P, Iolascon A, Albergoni MP, Basso G, Tonini GP. Department of Experimental Pathology, University of Bologna, Italy. Deletion of chromosome 1p and MYCN amplification have been reported as frequent abnormalities in human neuroblastoma. We studied loss of heterozygosity (LOH) in 50 (48 informative) Italian neuroblastoma patients by restriction fragment length polymorphisms (RFLPs) analysis using anonymous and hypervariable region (HVR) sequences. Twelve cases (25%) showed LOH at one or more loci. Locus D1S94 was the most frequently involved in LOH events (8/12) of deleted cases (66.6%). MYCN amplification was observed in 20% of patients which showed a significantly lower event-free survival probability (EFSp) (P = 0.004). We also studied the allelic distribution in the constitutional DNA of neuroblastoma patients (n = 44) and a matched group of healthy Italian subjects (n = 79) for loci D1S112 and D1S94. A significantly (P = 0.01) different allele frequency was detected for the two groups at locus D1S94, but not at D1S112. Moreover, the neuroblastoma population did not confirm the Hardy-Weinberg expectations at the former locus. This observation suggests the existence of an allelotype associated with neuroblastoma susceptibility. PMID: 9516830 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 205: Eur J Cancer. 1997 Oct;33(12):1937-41. Expression of a 260 kDa neuroblastoma surface antigen, the target of cytotoxic natural human IgM: correlation to MYCN amplification and effects of retinoic acid. David K, Ehrhardt A, Ollert MW, Erttmann R, Bredehorst R, Vogel CW. Department of Biochemistry and Molecular Biology, University of Hamburg, Germany. Human neuroblastoma cells contain a 260 kDa surface-associated antigen (NB-p260) that is recognised by natural cytotoxic IgM antibodies. In this study we demonstrate that NB-p260 is expressed in vivo in a neuroblastoma tumour specimen but not in normal human tissues of neuronal origin. Since MYCN amplification is a clinical marker of neuroblastoma disease progression, we analysed the expression of NB-p260 in human neuroblastoma cell lines with different MYCN amplification status. However, both amplified and non-amplified neuroblastoma cell lines exhibited comparable NB-p260 expression. Treatment of neuroblastoma cells with the differentiation-inducing agent retinoic acid (RA) also had no effect on the expression of NB-p260. Collectively, the data suggest that expression of NB-p260 on human neuroblastoma cells is independent of malignancy and differentiation status of neuroblastoma. PMID: 9516828 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 206: Eur J Cancer. 1997 Oct;33(12):1917-22. Loss of chromosome 1p may have a prognostic value in localised neuroblastoma: results of the French NBL 90 Study. Neuroblastoma Study Group of the Societe Francaise d'Oncologie Pediatrique (SFOP). Rubie H, Delattre O, Hartmann O, Combaret V, Michon J, Benard J, Peyroulet MC, Plantaz D, Coze C, Chastagner P, Baranzelli MC, Frappaz D, Lemerle J, Sommelet D. Unite d'Oncologie Pediatrique, CHU Purpan, Toulouse, France. Between March 1990 and December 1994, 316 consecutive children with localised neuroblastoma were registered in the French NBL 90 study. In addition to the assessment of a new chemotherapy regimen in unresectable neuroblastoma, we evaluated the prognostic significance of MYCN amplification and loss of the short arm of chromosome 1 (LOH1p). MYCN was found in 22/225 children (10%) and associated with unfavourable clinical features such as age at diagnosis > 1 year and large and unresectable tumours. LOH1p was observed in 9/91 patients (10%), of whom some had favourable prognostic factors such as age at diagnosis < 1 year (n = 4), INSS stage 1 or 2 (n = 3) and no MYCN amplification (n = 4). Overall survival (OS) and event-free survival (EFS) were, respectively, 56% and 22% (median follow-up: 36 months) for children with LOH1p compared with 97% and 94% for those without (log-rank = 10(-8)). All except 1 of the 5 children with MYCN amplification and LOH1p relapsed and ultimately died of the disease. Among the 4 with LOH1p and no MYCN amplification, recurrence occurred in 3 (2 local, 1 metastatic), all alive in second remission after salvage therapy (12-19 months after the relapse). In multivariate analysis, LOH1p was the strongest prognostic indicator for subsequent relapse. LOH1p appears more discriminant than MYCN amplification for predicting the risk of recurrence in children with localised neuroblastoma. However, its analysis was possible in only 30% of our patients and its final impact on survival should be confirmed in larger, prospective studies in order to stratify subsequent treatment. PMID: 9516824 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 207: Eur J Cancer. 1997 Oct;33(12):1911-6. Evidence that the MYCN oncogene regulates MRP gene expression in neuroblastoma. Norris MD, Bordow SB, Haber PS, Marshall GM, Kavallaris M, Madafiglio J, Cohn SL, Salwen H, Schmidt ML, Hipfner DR, Cole SP, Deeley RG, Haber M. Children's Leukaemia and Cancer Research Centre, Sydney Children's Hospital, Randwick, N.S.W., Australia. We have recently shown that expression of the multidrug resistance-associated protein (MRP) gene is a powerful prognostic indicator in childhood neuroblastoma and have suggested that the MYCN oncogene may regulate MRP gene expression. To address this hypothesis, we have examined the relationship between MYCN and MRP gene expression in neuroblastoma tumours and cell lines. MYCN and MRP gene expression were highly correlated in 60 primary untreated tumours both with (P = 0.01) and without MYCN gene amplification (P < 0.0001). Like MRP, high MYCN gene expression was significantly associated with reduced survival, both in the overall study population and in older children without MYCN gene amplification (relative hazards = 13.33 and 19.61, respectively). Inhibition of MYCN, through the introduction of MYCN antisense RNA constructs into human neuroblastoma cells in vitro, resulted in decreased MRP gene expression, determined both by RNA-PCR and Western analysis. The data are consistent with MYCN influencing neuroblastoma outcome by regulating MRP gene expression. PMID: 9516823 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 208: Oncol Res. 1997;9(9):467-76. Cell lineage and differentiation state are primary determinants of MYCN gene expression and malignant potential in human neuroblastoma cells. Spengler BA, Lazarova DL, Ross RA, Biedler JL. Department of Biological Sciences, Fordham University, Bronx, NY 10458, USA. Neuroblastoma cell lines typically exhibit multiple cell phenotypes, counterparts of those comprising the embryonic neural crest. Expression of the MYCN gene, usually amplified in cell lines, differs markedly among the various differentiation phenotypes. Whereas neuroblastic (N-type) and stem cell (I-type) sublines have abundant MYCN RNA and protein, S-type cells (nonneuronal neural crest precursors) have a 4- to 9-fold lower level of cytoplasmic mRNA and a 2- to 36-fold lower protein content. N and S sublines with chromosomally integrated MYCN genes have similar gene copy numbers. Thus, in these S cells, MYCN expression is downregulated. Nuclear run-on and mRNA stability assays have revealed similar transcription rates and mRNA half-lives in N and S cells from two cell lines, indicating that downregulation occurs posttranscriptionally prior to mRNA degradation in the cytoplasm. S-type cells derived from double minute chromosome-containing lines show 4- to 10-fold lower gene copy numbers than N counterparts. Experimental induction of differentiation to neuronal/neuroendocrine or to S-type cells results in a marked reduction of MYCNexpression and, in double minute chromosome-containing N-type sublines, in gene loss as well. Malignant potential as indicated by soft agar growth capacity and tumor formation in nude mice is markedly diminished in S cells and, generally, is directly proportional to MYCN mRNA levels. The most plausible relationship suggested by our data is that MYCN expression, regulated by cell lineage and/or differentiation state, directly modulates the malignant potential of human neuroblastoma cells. PMID: 9495452 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 209: Eur J Cancer. 1997 Sep;33(10):1627-33. Gain of chromosome arm 17q predicts unfavourable outcome in neuroblastoma patients. U.K. Children's Cancer Study Group and the U.K. Cancer Cytogenetics Group. Lastowska M, Cotterill S, Pearson AD, Roberts P, McGuckin A, Lewis I, Bown N. Department of Human Genetics, University of Newcastle upon Tyne, U.K. Gain of chromosome arm 17q has recently been reported in neuroblastoma tumours. We analysed 17q status in relation to other known prognostic features and clinical outcome in a series of 45 tumours. Chromosome 17 status was detected by cytogenetic analysis, fluorescence in situ hybridisation (FISH) anc comparative genomic hybridisation (CGH) and correlated with other clinical and genetic factors. Survival analysis was calculated by the Kaplan-Meier estimation. Twenty-eight out of 45 tumours showed 17q gain, and this was associated with established indicators of poor prognosis; stage 4 disease (P < 0.001), age above 1 year at diagnosis (P < 0.001), 1p deletion (P < 0.01), MYCN amplification (P = 0.03) and diploidy/tetraploidy (P = 0.04). 17q gain was associated with poor outcome: 3-year survival was 13.5% compared with 100% for tumours without 17q gain (P = 0.0001); and progression-free survival (PFS) was 8.1% after 3 years compared with 83% for 17q normal tumours (P = 0.0001). PFS in 28 MYCN non-amplified patients indicated that 17q status has discriminatory power within this group: PFS 0% for 17q gain (n = 14) versus 100% for normal 17q (n = 14) (P = 0.0001). This study indicates that 17q changes have prognostic significance in neuroblastoma and should be a target for molecular cytogenetic detection at diagnosis. PMID: 9389925 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 210: Virchows Arch. 1997 Oct;431(4):249-56. Characteristics of rhabdomyosarcoma cell lines derived from uterine carcinosarcomas. Emoto M, Iwasaki H, Oshima K, Kikuchi M, Kaneko Y, Kawarabayashi T. Department of Obstetrics and Gynecology, Fukuoka University School of Medicine, Japan. Rhabdomyosarcoma (RMS) is occasionally found in the female genital tract, and mostly appears as one of the heterologous mesenchymal components in uterine carcinosarcoma designated as malignant mixed mullerian tumour (MMMT). We examined the biological properties of a pure rhabdomyosarcoma (RMS) cell line designated FU-MMT-3, which was newly established from a surgical specimen taken from a patient with uterine MMMT. We also evaluated c-myc and MYCN gene amplification in three RMS cell lines (including FU-MMT-3) derived from three MMMTs by Southern blot analysis. FU-MMT-3 cells were propagated continuously for 57 serial passages over a 2-year period in vitro. FU-MMT-3 was able to produce tumours demonstrating pure RMS in athymic nude mice. Cytogenetically, FU-MMT-3 showed a triploidy pattern, with complex karyotypic abnormalities including trisomy of chromosome 8. All three RMS cell lines, including FU-MMT-3, showed amplification of the c-myc gene (approximately fourfold to eightfold), while no cell lines demonstrated MYCN gene amplification. FU-MMT-3 is considered to provide a useful system for the study of the biological behaviour of RMS in MMMTs. Extra copies of chromosome 8 and c-myc gene amplification may be associated with the rhabdomyoblastic differentiation in MMMT. Publication Types: Case Reports PMID: 9368662 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 211: Genes Chromosomes Cancer. 1997 Nov;20(3):243-52. Relational mapping of MYCN and DDXI in band 2p24 and analysis of amplicon arrays in double minute chromosomes and homogeneously staining regions by use of free chromatin FISH. Pandita A, Godbout R, Zielenska M, Thorner P, Bayani J, Squire JA. Department of Medical Biophysics, University of Toronto, Ontario, Canada. MYCN amplification has been observed in diverse neuronal tumors including neuroblastoma, retinoblastoma, and small cell carcinoma of the lung, and has been correlated with a poor prognosis in advanced-stage neuroblastomas. Recent studies have shown a co-amplification of DDXI, a DEAD box gene, and MYCN in retinoblastoma and neuroblastoma. DDXI has been mapped to within a megabase of the MYCN gene in band 2p24. In the present study, the relational map of DDXI and MYCN by fluorescence in situ hybridization (FISH) mapping to metaphase cells and extended free chromatin fibers indicated that DDXI is telomeric to MYCN. Dual-color FISH analysis of amplicons within arrays of extended chromatin fibers was performed to examine the physical relationship of MYCN and DDXI within double minute chromosomes (dmins) and homogeneously staining regions (hsrs). No regular reiterated amplicon repeat unit was present in the hsrs, but detailed analysis of the configurations of DDXI and MYCN within each array indicated that multiple rearrangements generated a complex hsr amplicon structure. Similarly, analysis of a cell line bearing dmins showed that a composite amplicon structure involving deletions and/or duplications of MYCN and DDXI is a feature of dmin formation. These data are consistent with a molecular mechanism involving many rearrangements during the evolution of gene amplification, resulting in complex amplicon structures with distinct changes in relative gene copy number and considerable variation in intragenic distances between coamplified genes. PMID: 9365831 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 212: Pediatr Pathol Lab Med. 1997 Nov-Dec;17(6):875-83. Heterogeneity of MYCN amplification in a child with stroma-rich neuroblastoma (ganglioneuroblastoma). Lorenzana AN, Zielenska M, Thorner P, Gerrie B, Weitzman S, Squire J. Department of Pediatrics, Hospital for Sick Children, Toronto, Ontario, Canada. Amplification of MYCN portends rapid tumor progression and poor prognosis in neuroblastoma. MYCN copy number has been described as homogeneous within a tumor and congruent in primary tumor and metastasis. We report a child with stage III favorable histology stroma-rich neuroblastoma (ganglioneuroblastoma) and a poor outcome with an apparent change in MYCN gene amplification by Southern blot. Initial biopsy revealed a ganglioneuroblastoma with predominance of differentiating cells designated as neuroblastoma, stroma-rich, intermixed (Shimada). Southern blot failed to demonstrate MYCN gene amplification. After front-line chemotherapy failed, a total resection was performed. In this specimen, Southern blot demonstrated MYCN amplification (15-20 copies) in the undifferentiated component and no amplification in the differentiated. Fluorescence in situ hybridization (FISH) analysis performed retrospectively on both tumor biopsies demonstrated MYCN amplification in the undifferentiated sections of both tumor specimens but not in the differentiated ones. This is the first well-documented case report of heterogeneous MYCN amplification in a child with neuroblastoma. Because key therapeutic decisions are based on the presence of MYCN amplification, physicians diagnosing and treating children with neuroblastoma need to be aware of the possibility that MYCN amplification may be heterogeneous within a tumor and may be missed using techniques based on pooled DNA such as Southern blotting. FISH may be a preferable method for determining MYCN amplification. Publication Types: Case Reports PMID: 9353827 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 213: Cancer Genet Cytogenet. 1997 Sep;97(2):135-42. Comparative genomic hybridization analysis of human neuroblastomas: detection of distal 1p deletions and further molecular genetic characterization of neuroblastoma cell lines. Van Roy N, Jauch A, Van Gele M, Laureys G, Versteeg R, De Paepe A, Cremer T, Speleman F. Department of Medical Genetics, University Hospital, Ghent, Belgium. Deletions of the short arm of chromosome 1 and MYCN amplification are the most frequently encountered genetic changes in disseminated neuroblastomas and neuroblastoma cell lines. Different strategies have been followed for detection of these and other genomic changes in neuroblastoma including karyotyping, FISH, and LOH, each with its own limitations. Here we report upon the evaluation of comparative genomic hybridization (CGH) in the analysis of neuroblastoma cell lines, with the emphasis on the assessment of the reliability of CGH for the detection of distal 1p deletions. We have analyzed seven neuroblastoma cell lines for which the 1p status was previously studied in detail using FISH and LOH. Our results show that CGH allows reliable detection of distal 1p deletions, including a small interstitial deletion in cell line SK-N-AS. Furthermore, CGH also allows the detection of chromosomal imbalance which would otherwise remain undetected, and provides useful information for further molecular characterization of chromosomal imbalances. Publication Types: Case Reports PMID: 9283597 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 214: Med Pediatr Oncol. 1997 Sep;29(3):206-7. Concomitant p53 mutation and MYCN amplification in neuroblastoma. Manhani R, Cristofani LM, Odone Filho V, Bendit I. Research and Molecular Biology Division, Pro-Sangue Hemocentro de Sao Paulo Foundation, Brazil. The MYCN oncogene is amplified in 20% of childhood neuroblastoma and is associated independently with poor prognosis. Alteration of the p53 tumor supressor gene, in contrast, occurs infrequently in these tumors. In this report, we described a 3-year-old girl with stage IV neuroblastoma. Molecular analysis revealed, both MYCN gene amplification and a point mutation of the p53 tumor supressor gene. To our knowledge, this is the first reported case of neuroblastoma with genetic alterations of both these genes. Publication Types: Case Reports PMID: 9212845 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 215: Med Pediatr Oncol. 1997 Aug;29(2):135-8. Application of fluorescence in situ hybridization to detect N-myc (MYCN) gene amplification on paraffin-embedded tissue sections of neuroblastomas. Hachitanda Y, Saito M, Mori T, Hamazaki M. Department of Pathology, National Children's Hospital, Tokyo, Japan. Fluorescence in situ hybridization (FISH) was applied to neuroblastoma for detection of N-myc (MYCN) oncogene amplification, and the results were compared with Southern blot analysis (Southern). In nine neuroblastomas (formalin-fixed paraffin-embedded tissues were available in seven cases including two cases with touch preparations, and two cell lines), all five cases with N-myc amplification detected by Southern had cells with multiple N-myc signals by FISH, and three cases showed no N-myc amplification either by Southern or FISH procedure. One case, not examined by Southern, showed amplified signals of N-myc by FISH. These data indicate that FISH results for N-myc amplification have close correlation with Southern blot analysis. The chromosome 2-specific repetitive DNA probe was also applied for the analysis of ploidy by FISH. Six cases with N-myc amplification by Southern and/or FISH had diploid tumors and two cases without amplified N-myc showed aneuploidy. The remaining one case consisted of heterogeneous elements showing diploidy in undifferentiated tissue and both aneuploidy (ganglionic cells) and diploidy (Schwann cells) in differentiated area. We conclude that FISH is a practical, useful and reliable method over Southern especially for analysis of N-myc amplification in neuroblastoma, and simultaneous cohybridization with a specific chromosome probe is of great value in predicting the prognosis of patients. PMID: 9180916 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 216: EMBO J. 1997 Jun 2;16(11):2985-95. Targeted expression of MYCN causes neuroblastoma in transgenic mice. Weiss WA, Aldape K, Mohapatra G, Feuerstein BG, Bishop JM. G.W. Hooper Foundation, and Department of Neurology, University of California, San Francisco 94143-0552, USA. The proto-oncogene MYCN is often amplified in human neuroblastomas. The assumption that the amplification contributes to tumorigenesis has never been tested directly. We have created transgenic mice that overexpress MYCN in neuroectodermal cells and develop neuroblastoma. Analysis of tumors by comparative genomic hybridization revealed gains and losses of at least seven chromosomal regions, all of which are syntenic with comparable abnormalities detected in human neuroblastomas. In addition, we have shown that increases in MYCN dosage or deficiencies in either of the tumor suppressor genes NF1 or RB1 can augment tumorigenesis by the transgene. Our results provide direct evidence that MYCN can contribute to the genesis of neuroblastoma, suggest that the genetic events involved in the genesis of neuroblastoma can be tumorigenic in more than one chronological sequence, and offer a model for further study of the pathogenesis and therapy of neuroblastoma. PMID: 9214616 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 217: Cancer. 1997 May 15;79(10):2028-35. Neuroblastoma in adults and adolescents: an indolent course with poor survival. Franks LM, Bollen A, Seeger RC, Stram DO, Matthay KK. Department of Pediatrics, University of California, San Francisco 94143-0106, USA. BACKGROUND: Neuroblastoma rarely occurs in adults, and less than 10% of cases occur in patients older than 10 years. It has been suggested that the behavior of this disease may be different in older patients than in young children. The purpose of this study was to investigate the presentation, biologic features, and outcome of adolescent and adult patients with neuroblastoma to define differences from childhood neuroblastoma. METHODS: Medical record and pathology reviews were conducted for 16 patients age 13 years or older (13-33 years) at diagnosis who presented with neuroblastoma at the University of California-San Francisco (UCSF) during the period 1968-1995 (patients with intracerebral tumors, esthesioneuroblastoma, or ganglioneuroma were excluded). Six of these patients received their original diagnosis at UCSF, and the others were referred after diagnosis. The survival for the same period for all neuroblastoma patients ages 13-18 years (n = 38) registered in the Children's Cancer Group (CCG) was compared with the survival for those ages 1-13 years (n = 1912). Three of the UCSF patients were enrolled in CCG studies. RESULTS: Biologic characteristics observed in adolescents and adults differed from those observed in younger patients. In the UCSF population, only 6 of 15 tested patients age 13 or older had elevated urinary catecholamines, and 0 of 6 tested patients had MYCN amplification. There were two patients with Stage I disease, three with Stage II, two with Stage III, and nine with Stage IV. Primary sites were adrenal, pelvic, and retroperitoneal in four cases each; mediastinal in two cases; and paraspinal in two cases. Metastases in nine patients at diagnosis were observed in bone in five; in bone marrow in four; in lymph nodes in three; in the liver in two; and in the pleura, breast, and dura in one patient each. 131I-metaiodobenzylguanidine uptake was observed in 9 of 11 patients. Initial treatment included surgery for 13 of 16 patients, chemotherapy for 10 of 16, radiation therapy for 7 of 16, and autologous bone marrow transplantation for 1 of 16. Relapses occurred in 15 of 16 patients and death in 13 of 16, with overall survival 30% 5 years after diagnosis. Only 1 patient currently remains free of clinical disease 24 months after diagnosis. Several of these patients had long courses from diagnosis to death, with multiple recurrences and/or chronic, persistent disease. In the CCG data base, 76% of patients ages 13-18 years had metastatic disease at diagnosis. In this group, only 1 of 32 had MYCN amplification. The actuarial survival of all CCG patients ages 13-18 years was 7% at 5 years and 4% at 10 years, whereas that for patients ages 1-13 years was 30% at 5 years and 23% at 10 years. CONCLUSIONS: Neuroblastoma in adolescents and adults has different biologic characteristics and a longer course than in children; nevertheless, ultimately the outcome is poor regardless of stage. A much more aggressive or innovative therapeutic approach is needed for these patients. PMID: 9149032 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 218: Genes Chromosomes Cancer. 1997 Mar;18(3):162-9. Comparative genomic hybridization study of primary neuroblastoma tumors. United Kingdom Children's Cancer Study Group. Lastowska M, Nacheva E, McGuckin A, Curtis A, Grace C, Pearson A, Bown N. Department of Human Genetics, University of Newcastle upon Tyne, UK. Neuroblastoma tumors show a complex interaction of genetic abnormalities, among which some are of significant prognostic importance; however, analysis of chromosome changes in this tumor is often unsuccessful. Twenty primary tumors were studied by comparative genomic hybridization (CGH), and abnormalities were found in 19. While these changes included deletions of chromosome arm Ip (45%) and MYCN oncogene amplification (30%), gains of chromosome 17 material were much more frequent (75%). We also found evidence in two cases of a new amplification site at band 2p23. PMID: 9071568 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 219: Int J Cancer. 1997 Feb 7;70(4):430-6. Pleiotropic over-expression of multidrug-resistance-related genes is correlated to MYCN and max mRNA accumulation during tumour progression in the IGR-N-91 human neuroblastoma model. Cappellen D, Benard J. Laboratory of Clinical and Molecular Pharmacology, Institut Gustave Roussy, Villejuif, France. An experimental model of advanced human neuroblastoma, IGR-N-91, which is able to disseminate in the nude mouse, has been described. The present study was designed to ascertain which cell population from the IGR-N-91 primary tumour actually disseminates throughout the animals. In s.c. IGR-N-91 tumour xenografts, 3 areas, called pearly, vascularized and haemorrhagic, depending on the presence of blood vessels and haemorrhagic suffusions, were consistently observed and independently resected. Molecular analysis of tumour materials revealed a significant increase in MYCN and max gene transcript levels in the haemorrhagic area, as compared with the pearly and vascularized areas. Given the growth kinetics observed both in vitro and in vivo, and the DNA flow-cytometry profiles of tumour cells obtained from the haemorrhagic area, this transcriptional increase did not appear to be associated with enhanced proliferation. In this area of the tumours, multidrug-resistance-related genes, i.e., MDRI, MRP, GST-pi and topoisomerase II alpha were activated concomitantly with MYCN and max genes. The same observations were made, except for the topoisomerase-II alpha gene, when sub-lines derived from metastases were compared with that derived from the primary tumour. These data demonstrate that over-expression of several genes determining the multi-drug-resistance phenotype precedes the metastatic spread of IGR-N-91 NB tumour cells in the nude mouse. Data also suggest that the cell sub-population exhibiting this pleiotropic over-expression within the primary tumour undergoes selection during metastatic dissemination. PMID: 9033651 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 220: Am J Pathol. 1997 Jan;150(1):81-9. Gain of chromosome 17 is the most frequent abnormality detected in neuroblastoma by comparative genomic hybridization. Plantaz D, Mohapatra G, Matthay KK, Pellarin M, Seeger RC, Feuerstein BG. Department of Laboratory Medicine, Division of Molecular Cytometry, University of California, San Francisco 94143-0808, USA. Neuroblastoma behavior is variable and outcome partially depends on genetic factors. However, tumors that lack high-risk factors such as MYCN amplification or 1p deletion may progress, possibly due to other genetic aberrations. Comparative genomic hybridization summarizes DNA copy number abnormalities in a tumor by mapping them to their positions on normal metaphase chromosomes. We analyzed 29 tumors from nearly equal proportions of children with stage I, II, III, IV, and IV-S disease by comparative genomic hybridization. We found two classes of copy number abnormalities: whole chromosome and partial chromosome. Whole chromosome losses were frequent at 11, 14, and X. The most frequent partial chromosome losses were on 1p and 11q. Gains were most frequent on chromosome 17 (72% of cases). The two patterns of gain for this chromosome were whole 17 gain and 17q gain, with 17q21-qter as a minimal common region of gain. Other common gains were on chromosomes 7, 6, and 18. High level amplifications were detected at 2p23-25 (MYCN region), at 4q33-35, and at 6p11-22. Chromosome 17q gains were associated with 1p and/or 11q deletions and advanced stage. The high frequency of chromosome 17 gain and its association with bad prognostic factors suggest an important role for this chromosome in the development of neuroblastoma. PMID: 9006325 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 221: J Clin Oncol. 1997 Jan;15(1):85-93. MYCN oncogene amplification in neuroblastoma is associated with worse prognosis, except in stage 4s: the Italian experience with 295 children. Tonini GP, Boni L, Pession A, Rogers D, Iolascon A, Basso G, Cordero di Montezemolo L, Casale F, Pession A, Perri P, Mazzocco K, Scaruffi P, Lo Cunsolo C, Marchese N, Milanaccio C, Conte M, Bruzzi P, De Bernardi B. Department of Hematology-Oncology, Giannina Gaslini Children's Hospital, Genova, Italy. tonini@cba.uniqe.it PURPOSE: To evaluate the prognostic role of MYCN oncogene amplification in children with neuroblastoma. PATIENTS AND METHODS: Of 694 children (age, 0 to 15 years) with previously untreated neuroblastoma, 295 (42%) were evaluated at diagnosis for MYCN gene amplification. RESULTS: Clinical characteristics and survival results of 295 patients studied and 399 not studied for MYCN were comparable. In 48 of 295 patients studied for MYCN (16%), the gene was amplified (> or = three gene copies). Amplification was more frequent in children older than 1 year, with abdominal tumor (18% v 7%), advanced disease, normal vanillylmandelic (VMA) urinary excretion, and high lactate dehydrogenase (LDH), ferritin, and neuron-specific enolase (NSE) serum levels. In patients studied for MYCN, the 5-year overall survival (OS) rate was higher for children aged less than 1 year (90% v 44%), with extraabdominal tumor, stage 1 or 2 versus 3 versus 4, and normal NSE, LDH, and ferritin serum levels. Patients with amplified MYCN had a worse OS (odds ratio [OR], 3.38; confidence interval [CI], 2.22 to 5.16). This association held after adjustment for other characteristics. The impact of MYCN amplification was greater in patients with favorable characteristics, in particular age (OR, 10.28 for infants; 2.08 for older children) and stage (OR, 35.3 for stage 1 to 2; 8.41 for stage 3; 1.76 for stage 4). However, of 29 children with stage 4s, all three with amplified MYCN survive. In a multivariate analysis, the prognostic role of MYCN amplification, age, and stage was confirmed, but the size of the effect of MYCN was dependent on age and stage. CONCLUSION: MYCN amplification is associated with a worse prognosis in children with neuroblastoma at all ages and stages except 4s. This association is most pronounced in children with otherwise favorable prognostic indicators, and in these children should be considered as an indication for more intensive intervention. PMID: 8996128 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 222: Oncogene. 1996 Oct 3;13(7):1561-5. Physical mapping of the DDX1 gene to 340 kb 5' of MYCN. Kuroda H, White PS, Sulman EP, Manohar CF, Reiter JL, Cohn SL, Brodeur GM. Division of Oncology, Children's Hospital of Philadelphia, Pennsylvania, USA. One of the most important prognostic factors in neuroblastoma is amplification of the MYCN gene, which is strongly associated with advanced stages of disease and a poor prognosis. Although the MYCN amplicon sometimes spans more than 1 Mb, no other consistently expressed sequences from the MYCN amplicon have been reported. However, DDX1, a gene encoding a DEAD box protein, was recently mapped to chromosome 2p24 and is frequently co-amplified with MYCN. Therefore, we performed genomic mapping with YACs to determine the physical relationship between DDX1 and MYCN, and whether DDX1 was contained within the core region of amplification. Based on YAC restriction mapping and content analysis, DDX1 maps 340 kb 5' of MYCN, outside the core domain of consistent amplification. Interestingly, we also determined by sequence analysis and detailed restriction mapping that G21, previously isolated as a 'neuroblastoma-specific' cDNA clone from an MYCN amplicon, is a partial cDNA of DDX1. Our data confirm that DDX1 is amplified in some but not all MYCN-amplified tumors, and that it is rearranged in other cases. This suggests that the co-amplification of DDX1 is due to its proximity to MYCN. PMID: 8875996 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 223: Oncogene. 1996 Sep 5;13(5):1065-72. Human glioblastomas with no alterations of the CDKN2A (p16INK4A, MTS1) and CDK4 genes have frequent mutations of the retinoblastoma gene. Ichimura K, Schmidt EE, Goike HM, Collins VP. Institute for Oncology and Pathology, Karolinska Hospital, Stockholm, Sweden. A series of 195 human gliomas were studied as to the status of their CDKN2A, CDK4 and RB1 genes. Among 120 glioblastomas, 40% had no wild-type CDKN2A gene, 12% amplified the CDK4 gene, and 14% had no wild-type RBI gene. With two exceptions, each tumour had only one of these abnormalities. Thus the majority of the glioblastomas (64%) had distinct genetic aberrations which would obviously disrupt the control of transition from G1 to the S-phase of the cell cycle. A further 30% had loss of one allele of the CDKN2A and/or RBI genes. Only seven (6%) glioblastomas had no abnormalities of these genes. Anaplastic astrocytomas showed similar changes to the glioblastomas but at lower frequencies-34% showing no aberrations of the genes analysed. The astrocytomas showed solely loss of one allele of the RBI gene in 28% of tumours, with retention of one wild-type copy. In the glioblastomas with no alterations of CDKN2A, CDK4 or RB1, several other genes (CCND1, CCND2, CCND3, CDK6, E2F, CDK7, MYC and MYCN) whose products take part in cell cycle regulation were examined. No abnormalities were detected. Thus some aberration of the CDKN2A, CDK4 and RB1 genes appears to be almost obligatory in glioblastomas. PMID: 8806696 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 224: Br J Cancer. 1996 Sep;74(5):773-9. Expression of mRNA for the neurotrophin receptor trkC in neuroblastomas with favourable tumour stage and good prognosis. Ryden M, Sehgal R, Dominici C, Schilling FH, Ibanez CF, Kogner P. Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden. Childhood neuroblastoma tumours of the sympathetic nervous system show a remarkable clinical heterogeneity ranging from spontaneous regression to unfavourable outcome despite intensive therapy. Favourable neuroblastomas often express high levels of trkA mRNA, encoding the tyrosine kinase receptor for nerve growth factor. We have investigated mRNA expression for the neurotrophin receptor trkC in 23 primary neuroblastomas using a sensitive RNAase protection assay. TrkC expression was detected in 19 of these tumours at highly variable levels with a 300-fold difference between the highest and lowest values. Significantly higher levels of trkC mRNA were found in tumours from patients with favourable features such as low age (P < 0.012), favourable tumour stage (P < 0.012) and favourable prognosis (P < 0.05). Children with intermediate or high trkC mRNA expression had better prognosis compared with those with low or undetectable levels (83.3% vs 20%, P = 0.005). Further characterisation of trkC mRNA expression by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that mRNA encoding the full-length cytoplasmic tyrosine kinase domain of the receptor was only expressed in a subset of favourable tumours. These data show that favourable neuroblastomas may express the full trkC receptor while advanced tumours, in particular MYCN-amplified neuroblastoma, seem to either express no trkC or truncated trkC receptors of as yet unknown biological function. These data are suggestive of a role for trkC and its preferred ligand neutotrophin-3, NT-3, in neuroblastoma differentiation and/or regression. PMID: 8795581 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 225: Genes Chromosomes Cancer. 1996 Jul;16(3):196-203. Mapping of chromosomal gains and losses in primitive neuroectodermal tumors by comparative genomic hybridization. Schutz BR, Scheurlen W, Krauss J, du Manoir S, Joos S, Bentz M, Lichter P. Deutsches Krebsforschungszentrum, Abteilung Organisation komplexer Genome, Heidelberg, Germany. A series of 18 primitive neuroectodermal tumors (PNETs), the most common malignant central nervous system tumors of childhood, were analyzed with the recently developed approach of comparative genomic hybridization (CGH). In five cases, in which only small amounts of DNA were available, universal polymerase chain reaction was successfully applied to generate adequate probe material. In 15 tumors, chromosomal imbalances were elicited, most frequently involving chromosome 17 (loss of 17p and gain of 17q). Further recurrent imbalances included gains of the distal regions of 4p, 5p, 5q, 7q, 8q, and 9p. High-level amplifications were found on 2p24 (one case) and 8q24 (three cases), suggesting involvement of the protooncogenes MYCN and MYC, respectively. In one of these cases, Southern blot analysis could be performed, proving high-copy-number amplification of MYC. Interestingly, none of the three patients with high-copy-number amplifications of MYC responded to therapy. PMID: 8814453 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 226: Genes Chromosomes Cancer. 1996 Feb;15(2):134-7. The DDX1 gene maps within 400 kbp 5' to MYCN and is frequently coamplified in human neuroblastoma. Amler LC, Schurmann J, Schwab M. Department of Cytogenetics, German Cancer Research Center, Heidelberg. Human neuroblastoma cells frequently show amplification of the oncogene MYCN, which maps to 2p24. Previous studies have localized the DEAD box motif gene DDX1 to the same chromosome band and demonstrated coamplification of DDX1 and MYCN in two retinoblastoma cell lines. Recently, a high frequency of coamplification of DDX1 and MYCN has been shown in human neuroblastoma cells. We have determined the physical distance between the two genes by pulsed field gel electrophoresis in normal tissue and have found that DDX1 maps to a position at a maximum distance of 400 kbp 5' to MYCN. Two neuroblastoma cell lines with coamplification of DDX1/MYCN showed a similar topographic relationship of the two genes. In contrast, in two cell lines with high copy number, the DDX1 gene was not present in all amplified units recognized by MYCN and had changed its position in the amplified DNA relative to MYCN from 5' to 3', presumably by rearrangement during the amplification process. Our data show that the high frequency of DDX1 coamplification is due to its close physical distance to MYCN. Although amplification has resulted in an elevated expression of DDX1 the significance of overexpression for neuroblastoma remains unclear. PMID: 8834178 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 227: Genes Chromosomes Cancer. 1996 Feb;15(2):129-33. Amplification of a DEAD box gene (DDX1) with the MYCN gene in neuroblastomas as a result of cosegregation of sequences flanking the MYCN locus. Noguchi T, Akiyama K, Yokoyama M, Kanda N, Matsunaga T, Nishi Y. Life Science Research Laboratory, Japan Tobacco, Inc., Kanagawa. A DEAD box gene (DDX1) characterized by a motif with a putative RNA helicase was found at elevated levels, with multiple copies, in a neuroblastoma and in some retinoblastoma cell lines in which the MYCN gene was amplified. The present study was aimed at determining whether amplification of the DDX1 gene is critical for human neuroblastomas exhibiting MYCN gene amplification. Extended DNA panels of tumors and cell lines revealed amplification of the DDX1 gene in approximately half of the specimens exhibiting MYCN gene amplification, which is in good agreement with a finding reported recently. Because its profile was similar to that of the cDNA marker G21 and another flanking DNA marker, clone 8, both of which localize outside the core of the amplicon of the MYCN gene, we noted that we could localize the DDX1 gene in relation to the MYCN gene. Utilizing pulsed-field gel electrophoresis according to a method based on the combinatorial alignment of multiple single digests and a 5.5-megabase map surrounding the MYCN locus, we mapped the DDX1 gene within a 100 kb region about 400 kb upstream from the MYCN gene, where G21 is localized. Further hybridization experiments with both genes, complete sequencing of G21, and its comparison with that of the DDX1 gene eventually confirmed that the DDX1 gene is identical to G21. G21 is a cDNA clone isolated by differential screening of a library from a neuroblastoma cell line, IMR-32, but its function has not yet been identified. Coamplification of the DDX1 gene with the MYCN gene is a consequence of the segregation of continuous DNA stretches spanning both loci during the amplification process. PMID: 8834177 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 228: Pediatr Pathol Lab Med. 1995 Nov-Dec;15(6):831-44. Application of a simplified comparative genomic hybridization technique to screen for gene amplification in pediatric solid tumors. Bayani J, Thorner P, Zielenska M, Pandita A, Beatty B, Squire JA. Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada. Conventional cytogenetic analysis of solid tumors is technically very demanding and requires a large number of viable cells. The technique of comparative genomic hybridization (CGH) circumvents these difficulties and has been shown to be particularly useful for identifying new gene amplifications. We have simplified the CGH technique for the detection of amplifications by utilizing a single labeling approach in which labeled tumor DNA is mixed with unlabeled normal human DNA and hybridized to normal metaphases on a slide. To examine the consistency and sensitivity of the method, initial experiments were performed using a retinoblastoma (RB) cell line and five pediatric solid tumors known to contain an amplification. The technique was easy to use and sensitive enough to detect low-level amplifications. The RB cell line showed reproducible signals at 2p24, indicative of amplified sequences, on both homologues in 95% of the metaphases (> 30) examined. Amplifications of the MYCN gene (2p24) were detected in three alveolar rhabdomyosarcomas and one medulloblastoma. CGH was then applied to six tumors in a prospective fashion, before data about specific gene amplification were available. In two, amplification of the MDM2 gene (12q13-14) was identified using CGH and later confirmed by Southern blot analysis. Four tumors negative for MDM2 and MYCN amplifications by CGH analysis were also negative by Southern blot analysis. Gene amplification as low as fourfold was detected in one tumor and the overall pattern of gene amplification detected by CGH in these tumors was not complex, involving just one amplification site for each case. Therefore, this simplified CGH technique is suitable for routine screening of pediatric solid tumors for amplifications when genetic studies are important but sample sizes are small and dividing cells are infrequent or unavailable. PMID: 8705194 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 229: Genes Chromosomes Cancer. 1995 Nov;14(3):196-203. Co-amplification and concomitant high levels of expression of a DEAD box gene with MYCN in human neuroblastoma. Manohar CF, Salwen HR, Brodeur GM, Cohn SL. Department of Pediatrics, Northwestern University, Chicago, Illinois, USA. MYCN gene amplification is strongly correlated with poor prognosis in neuroblastoma (NB), the second most common solid pediatric tumor. However, increased MYCN expression seen in tumors that lack MYCN amplification does not correlate with aggressive clinical behavior. Whereas the MYCN gene spans only 7 kb, the MYCN amplicon has been shown to range in size from 350 kb to more than 1 Mb. Given the large size of the amplicon, it is possible that additional genes are co-amplified in NBs whose expression may contribute to the aggressive phenotype associated with MYCN-amplified tumors. We isolated a cDNA clone from a human NB library that is identical to DDXI, a gene recently reported to be preferentially expressed in two retinoblastoma cell lines that also express high levels of MYCN. DDXI belongs to a family of genes that encode DEAD (Asp-Glu-Ala-Asp) box proteins, putative ATP-dependent RNA helicases implicated in a number of cellular processes involving alterations of RNA secondary structure. We examined the frequency of DDXI amplification in 15 NB cell lines, 1 neuroepithelioma cell line, and 122 NB tumors by Southern blot analyses, and we found that 7 of 10 MYCN-amplified cell lines and 27 of 40 (68%) MYCN-amplified tumors also harbored multiple copies of the DDXI gene. Amplification of DDXI was associated with high levels of DDXI mRNA expression in the NB cell lines and tumors as examined by Northern analysis. Neither DDXI gene amplification nor enhanced expression was observed in tumors or cell lines that lacked MYCN amplification. Because RNA helicases play important roles in both post-transcriptional and translational gene regulation, high levels of DDXI expression consequent to genomic amplification may contribute to the malignant phenotype of a subset of NBs. PMID: 8589036 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 230: Cancer Res. 1995 Oct 15;55(20):4664-9. Significance of chromosome 1p loss of heterozygosity in neuroblastoma. Maris JM, White PS, Beltinger CP, Sulman EP, Castleberry RP, Shuster JJ, Look AT, Brodeur GM. Division of Oncology, Children's Hospital of Philadelphia, University of Pennsylvania, Philadepphia School of Medicine 19104, USA. We analyzed 156 primary neuroblastoma tumor samples for loss of heterozygosity at the distal short arm of chromosome 1 (1p LOH). We also compared 1p LOH with known clinical and genetic prognostic variables as well as patient outcome. 1p LOH was detected in 30 of 156 tumors (19%) and was strongly associated with adverse clinical and biological features. 1p LOH was also strongly predictive of a poor outcome in univariate analyses (estimated 4-year survival, 32 +/- 10% SE versus 76 +/- 5% SE; P < 0.001). However, the prognostic value of 1p LOH was equivocal when stratified for amplification of the MYCN oncogene (P = 0.16). We conclude that 1p LOH is an important component of a pattern of genetic abnormalities in neuroblastoma associated with an aggressive clinical course. PMID: 7553646 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 231: J Natl Cancer Inst. 1995 Oct 4;87(19):1470-6. Identification of subsets of neuroblastomas by combined histopathologic and N-myc analysis. Shimada H, Stram DO, Chatten J, Joshi VV, Hachitanda Y, Brodeur GM, Lukens JN, Matthay KK, Seeger RC. Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles, CA 90027, USA. BACKGROUND: Neuroblastomas show different histopathologic phenotypes, and the tumor cells can carry normal or multiple copies of the N-myc proto-oncogene (MYCN). Studies of the N-myc gene and histopathology of untreated primary neuroblastomas have demonstrated that both these factors are important in risk assessment. PURPOSE: Our purpose was to determine if there are any associations between N-myc gene copy number, histopathologic features, clinical stage, and progression-free survival (PFS) and if joint analyses of histopathology and N-myc gene copy number improve risk assessment. METHODS: The histopathologic phenotype and N-myc gene copy number were determined for 232 biopsy/surgery specimens obtained from untreated primary neuroblastoma patients. Tumors were classified as having favorable or unfavorable histology on the basis of Schwannian stroma (rich versus poor), neuroblastic differentiation (differentiating versus undifferentiated), and mitosis-karyorrhexis (fragmenting nucleus) index (MKI; high, intermediate, or low) in the context of age at diagnosis (Shimada classification). N-myc gene amplification was considered significant when the gene copy number was at least 10-fold higher than normal as determined by Southern blot analysis. Otherwise, tumors were classified as nonamplified for N-myc. RESULTS: Among 19 stroma-rich tumors, 11 had grossly visible neuroblastic nodules, and two of these had N-myc amplification. Of 213 stroma-poor tumors, 51 had N-myc amplification, all of which were undifferentiated, and 45 (88% of 51) had high MKI. This histologic phenotype was present in less than 10% of tumors with nonamplified N-myc. Of 162 stroma-poor tumors that showed nonamplified N-myc, 45 (28%) were differentiating and 121 (75%) had low MKI. Neuroblastomas of clinical stages I, II, and IV-S nearly always had favorable histology and no amplification of N-myc. Stage III (regional) and particularly stage IV (metastatic) tumors, however, frequently had unfavorable histologic features with or without N-myc amplification. The estimated PFS at the end of 4 years after diagnosis was 83% for patients whose tumors had favorable histology and no N-myc amplification. The estimated PFS for the patients whose neuroblastomas had unfavorable histology, however, was 29% without and 13% with N-myc amplification, respectively. Subsets of patients with stage II, III, or IV disease were identified by both histologic evaluation and N-myc analysis. Multivariate Cox regression analysis indicated that both the histologic and N-myc-based stratifications provided prognostic information that was independent of staging. CONCLUSIONS: Neuroblastomas with N-myc amplification have a characteristic histopathologic phenotype and an aggressive clinical course. In contrast, neuroblastomas without N-myc amplification exhibit a wide range of histologic features that can define prognostic subsets. PMID: 7674334 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 232: Pediatr Pathol Lab Med. 1995 Sep-Oct;15(5):733-44. Molecular and cytogenetic analysis of a cerebellar primitive neuroectodermal tumor with prominent neuronal differentiation: detection of MYCN amplification by differential polymerase chain reaction and Southern blot analysis. Jay V, Squire J, Zielenska M, Gerrie B, Humphreys R. Department of Pathology, Hospital for Sick Children, University of Toronto, Ontario, Canada. Oncogene amplification is uncommon in cerebellar primitive neuroectodermal tumor (medulloblastoma) and its frequency and diversity are greater in medulloblastoma cell lines. We describe a medulloblastoma in a 10-year-old-girl with striking neuronal differentiation evident in the islands of ganglion cells intermixed with more primitive undifferentiated cells. The islands of ganglion cells showed prominent synaptophysin positivity. Karyotypic analysis revealed hypo- and hyperdiploidy with multiple random rearrangements and double minute chromosomes. Differential polymerase chain reaction and Southern blot analysis revealed up to 25-fold MYCN amplification. Publication Types: Case Reports PMID: 8597859 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 233: Genes Chromosomes Cancer. 1995 Sep;14(1):63-7. Mitochondrial ATP synthase alpha-subunit gene amplified in a retinoblastoma cell line maps to chromosome 18. Bie W, Squire JA, Fraser M, Paterson MC, Godbout R. Molecular Oncology Program, Cross Cancer Institute, Edmonton, Alberta, Canada. The human retinoblastoma cell line Y79 has multiple copies of the MYCN gene and the DEAD box gene DDXI. Both genes have been mapped to chromosome band 2p24. A third gene, encoding the alpha-subunit of mitochondrial ATP synthase (ATPSA), is also amplified in Y79. Here we report that there are at least four human mitochondrial ATPSA-related genes located on four different chromosomes. The ATPSA gene that is amplified in Y79 originates from chromosome 18. In Y79, the amplified copies of both the ATPSA and the MYCN genes are located on a homogeneously staining region (HSR) at chromosome band Ip34. PMID: 8527386 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 234: Leuk Lymphoma. 1995 Aug;18(5-6):511-4. Activation of MYCN in a case of non-Hodgkin's lymphoma. Finnegan MC, Hammond DW, Hancock BW, Goyns MH. Department of Clinical Oncology, Sheffield University Medical School, United Kingdom. We have examined a series of non-Hodgkin's lymphomas (NHL) for evidence of expression of the MYC gene family. Northern blot analysis of RNA samples derived from 11 non-malignant reactive lymphoid tissues and 33 NHL was used to investigate expression of MYC, MYCL and MYCN. As expected MYC expression was detected in all samples. The levels of MYC expression were quantified by densitometry and appeared to be 3-8 fold higher in high grade NHL than in the low grade NHL or non-malignant lymphoid tissue. No expression of MYCL was detected in any sample. Expression of MYCN was however observed in one sample, which had been diagnosed as a T-cell high grade NHL. A detailed cytogenetic analysis of this sample proved difficult to obtain but, by using fluorescence in-situ hybridization (FISH), we were able to demonstrate that on one of the chromosomes 2 the MYCN gene was localised to a translocation breakpoint region. It therefore appears that in NHL it is possible for MYCN, like MYC in Burkitt lymphoma, to be activated as a result of a chromosome translocation event. Publication Types: Case Reports PMID: 8528061 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 235: Diagn Mol Pathol. 1995 Jun;4(2):128-35. Fluorescence in situ hybridization (FISH) detection of MYCN oncogene amplification in neuroblastoma using paraffin-embedded tissues. Misra DN, Dickman PS, Yunis EJ. Department of Pathology, Children's Hospital of Pittsburgh, PA 15213, USA. The expression and degree of amplification of the MYCN oncogene in neuroblastoma provide an important indicator of disease prognosis. Detection of MYCN amplification has been described using Southern blotting or polymerase chain reaction (PCR) on DNA from fresh or frozen tissue samples, and using in situ hybridization mainly on metaphase spreads or smears of cultured neuroblastoma cells. In this article, we describe fluorescence in situ hybridization (FISH) results on detection of MYCN amplification in formalin-fixed, paraffin-embedded samples of 25 neuroblastoma and 20 nonneuroblastoma pediatric tumors. MYCN amplification was readily detectable by FISH in eight of the neuroblastomas; correlation with results obtained by Southern analysis was perfect. Of the nonneuroblastoma tumors, only one of three retinoblastoma cases showed MYCN amplification. In contrast to the Southern blot technique, FISH demonstrated the state of amplification heterogeneity of the tumor cells as well as the nature of the amplification units: double-minute chromosomes (DMs) or homogeneously staining regions (HSRs). The results indicate that FISH is a rapid and reliable method for detection of MYCN oncogene amplification in routinely processed samples and may be used to supplant the Southern blot technique. PMID: 7551293 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 236: Oncogene. 1995 Apr 6;10(7):1417-22. Co-amplification of MYCN and a DEAD box gene (DDX1) in primary neuroblastoma. Squire JA, Thorner PS, Weitzman S, Maggi JD, Dirks P, Doyle J, Hale M, Godbout R. Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada. DEAD box proteins are putative RNA helicases that have been implicated in cellular processes involving alteration of RNA secondary structure, such as translation initiation and splicing. These proteins share eight conserved amino acid motifs, including Asp(D)-Glu-(E)-Ala(A)-Asp(D) which is part of a more extended motif. Recently, we have shown that the novel DDX1 gene containing a DEAD box motif maps to the same chromosome band as MYCN at 2p24 and is co-amplified with MYCN in retinoblastoma cell lines. Here, we show that the DDX1 gene is co-amplified with the MYCN gene in 2 of three neuroblastoma cell lines and that DDX1 RNA levels correlate with DDX1 gene copy number. Since amplification of MYCN is an indicator of poor prognosis in neuroblastoma, it was of interest to determine whether co-amplification with DDX1 occurred in clinical samples of neuroblastoma and whether such a finding carried any additional prognostic significance. We determined the gene copy number of DDX1 in 32 neuroblastoma patient samples (representative of all stages): 13 were MYCN amplified and 19 had normal copy numbers of the MYCN gene. Of the 13 neuroblastomas that were MYCN amplified, seven were also DDX1 amplified. Of the 19 that were not MYCN amplified, none were DDX1 amplified. This is the first example of a gene that is co-amplified with MYCN at a high frequency in neuroblastoma. While there was a trend towards a worse clinical outcome with co-amplification, the numbers were too small to reach significance. Publication Types: Case Reports PMID: 7731693 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 237: Cancer. 1995 Apr 1;75(7):1694-9. Morphologic and molecular cytogenetics in neuroblastoma. Avet-Loiseau H, Venuat AM, Benard J, Leibovitch MP, Hartmann O, Bernheim A. Laboratoire de Cytogenetique et Genetique Oncologiques, Institut Gustave Roussy, France. BACKGROUND: Some genetic alterations have been shown to have prognostic implication for patients with neuroblastoma: MYCN oncogene amplification, deletion of the short arm of chromosome 1 and di- or tetraploidy. The goal of this study was to analyze these factors in children with neuroblastoma. METHODS: Twenty neuroblastoma samples were analyzed with morphologic cytogenetics, and each of them was compared with MYCN amplification status by Southern blot and fluorescent in situ hybridization (FISH) with a genomic probe. RESULTS: A complete karyotype was obtained for 14 children. A diploid or tetraploid mode and a 1p deletion were found in most children with advanced stages. MYCN amplification status was totally concordant with both methods in all patients, even in a case with low level amplification. A wide intercellular variation in the amplification level in each MYCN amplified sample was shown. CONCLUSION: The use of FISH to assess MYCN amplification rapidly in neuroblastoma is recommended. This method could be very useful in future therapeutic protocols in which treatment is based on MYCN status (and especially for infants and children with localized tumor). PMID: 8826929 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 238: Oncogene. 1995 Mar 16;10(6):1081-6. Non-syntenic amplification of MDM2 and MYCN in human neuroblastoma. Corvi R, Savelyeva L, Breit S, Wenzel A, Handgretinger R, Barak J, Oren M, Amler L, Schwab M. Division of Cytogenetics, German Cancer Research Center, Heidelberg. Amplification of the MYCN gene is a well documented genetic alteration of aggressively growing human neuroblastomas. Through cytogenetic studies we have identified neuroblastoma cell lines which, in addition to amplified MYCN, carry amplified DNA not harbouring MYCN. In situ hybridization of biotinylated total genomic DNA to metaphase chromosomes of normal human lymphocytes by reverse genomic hybridization revealed the amplified DNA to be derived from chromosome 12 band q13-14. Subsequent filter analyses showed a 20- to 40-fold amplification of the MDM2 gene, located at 12q13-14, both in three cell lines and in an original tumor, in addition to amplified MYCN. As the apparent consequence of amplification abundant MDM2 protein was present, a part of which was complexed with p53. PMID: 7700632 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 239: Eur J Cancer. 1995;31A(4):637-41. First experience with prognostic factors in unselected neuroblastoma patients. The Austrian Neuroblastoma 87 Study. Ladenstein R, Urban C, Gadner H, Fink FM, Zoubek A, Emminger W, Grienberger H, Schmitt K, Ambros PF, Ambros IM, et al. St Anna Children's Hospital, Vienna, Austria. Between January 1987 and December 1993, 117 patients were registered in the Austrian A-NB87 study. The male/female ratio was 1.18, with 50 patients below the age of 1 year at diagnosis. Patients were assigned to stage according to the result of primary surgery in localised disease. Age, ferritin and neuron specific enolase were used in addition in stage III disease for risk-adapted treatment. Adrenal or pelvic primary tumour sites were mainly associated (81%) with advanced disease. The median observation time of the study is 3.5 years. The overall survival at 3 years was excellent in low stage disease and IVs patients, i.e. 100% for stage I and IIA (20 patients), 92% in stage IVs (13 patients), 81% in stage IIIA (18 patients) and 69% in stage IIB (8 patients). Stage IV (38 patients) showed a survival rate of 51%, whereas stage IIB (10 patients) had the worst outcome in this study, i.e. 20%, due to treatment-related toxicity. Significant unfavourable prognostic factors were neuron specific enolase (NSE) > 100 ng/ml, ferritin > 300 micrograms/ml and amplified MYCN. This study achieved a better survival rate in stage IV patients and a subgroup of stage III in comparison to our previous study (Padiatrie und Padologie 1986, 21, 269) and gives the basis to further reduce treatment intensity in low-risk disease based on biological factors. However, prognosis for high-risk cases was still poor in spite of a very aggressive treatment concept. Publication Types: Clinical Trial PMID: 7576985 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 240: Eur J Cancer. 1995;31A(4):560-4. Genetic alterations associated with metastatic dissemination and chemoresistance in neuroblastoma. Benard J. Laboratory of Clinical & Molecular Pharmacology, Institut Gustave Roussy, Villejuif, France. Knowledge about genetic alterations specific to the metastatic process and chemoresistance in neuroblastoma is progressing steadily. Low or no CD44 expression, increased NM23 expression and specific mutations of the 5' coding regions of NM23 are distinct features of aggressive, metastatic neuroblastoma. MYCN down-regulates Class I HLA antigen expression in many neuroblastoma cell lines and, in turn, may be regulated by a suppressor gene. The MYCN amplified human neuroblastoma cell line, IGR-N-91, established in vitro, metastasises in the nude mouse and has exhibited co-activation of MYCN and PGY1, resulting from direct activation of the oncoprotein on the PGY1 promoter. In this model, the MYCN product activates angiogenesis, the dissemination process and chemoresistance via specific genes (PGY1 and GST3). MYCN, like the BCL-2 and TP53 products, may also play a key role in apoptosis. The implication of these genes in the potential for metastasis and chemoresistance in neuroblastoma is discussed. Publication Types: Review Review, Tutorial PMID: 7576968 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 241: Eur J Cancer. 1995;31A(4):545-9. Evaluation of CD44 prognostic value in neuroblastoma: comparison with the other prognostic factors. Combaret V, Lasset C, Frappaz D, Bouvier R, Thiesse P, Rebillard AC, Philip T, Favrot MC. Hopital Edouard Herriot, Lyon, France. CD44 gene products are potential markers of aggressiveness in different tumour models, a result which prompted us to study clinical neuroblastoma (NB) specimens. CD44 expression was determined by immunostaining of 52 tumour samples from newly diagnosed NB with a monoclonal antibody (J173) directed against an epitope common to all CD44 isoforms. CD44 immunoreactivity was detected in 37 of the tumours (71%). CD44 was expressed in all 22 NBs with favourable prognoses (stages 1, 2 or 4S), but only 50% (15/30) of advanced NB (stages 3 and 4) (P < 10(-4)), suggesting that the absence, rather than the overexpression, of CD44 is a signal of tumour aggressiveness. The cumulative progression-free survival was significantly longer in patients with CD44 positive tumours compared with patients with CD44 negative tumours (P < 10(-5)). More importantly, progression-free survival was also significantly higher in CD44 positive patients within the high-risk group (P < 0.01). In univariate analysis, we tested the prognostic value of tumour expression of CD44 in comparison with tumour stage, age, tumour histology, and presence or absence of amplification of the MYCN protooncogene. All five measures had significant prognostic value. The expression of CD44 and the absence of MYCN amplification were the most powerful predictors of a favourable outcome. In a multivariate analysis of these measures, CD44 expression and tumour stage were the only independent prognostic factors for the prediction of patient survival. NB is the first clinical model described in which tumour aggressiveness correlates with repression rather than stimulation of CD44 expression. We recommend the use of CD44 as an additional biological marker in the initial staging of NB. PMID: 7576964 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 242: Eur J Cancer. 1995;31A(4):541-4. Comparison of DNA aneuploidy, chromosome 1 abnormalities, MYCN amplification and CD44 expression as prognostic factors in neuroblastoma. Christiansen H, Sahin K, Berthold F, Hero B, Terpe HJ, Lampert F. Klinik fur Kinderheilkunde, Universitat zu Koln, Germany. A comparison of the prognostic impact of five molecular variables in a large series was made, including tests of their nonrandom association and multivariate analysis. Molecular data were available for 377 patients and MYCN amplification, cytogenetic chromosome 1p deletion, loss of chromosome 1p heterozygosity, DNA ploidy and CD44 expression were investigated. Their interdependence and influence on event-free survival was tested uni- and multivariately using Pearson's chi 2-test, Kaplan-Meier estimates, log rank tests and the Cox's regression model. MYCN amplification was present in 18% (58/322) of cases and predicted poorer prognosis in localised (P < 0.001), metastatic (P = 0.002) and even 4S (P = 0.040) disease. CD44 expression was found in 86% (127/148) of cases, and was a marker for favourable outcome in patients with neuroblastoma stages 1-3 (P = 0.003) and 4 (P = 0.017). Chromosome 1p deletion was cytogenetically detected in 51% (28/55), and indicated reduced event-free survival in localised neuroblastoma (P = 0.020). DNA ploidy and loss of heterozygosity on chromosome 1p were of less prognostic value. Most factors of prognostic significance were associated with each other. By multivariate analysis, MYCN was selected as the only relevant factor. Risk estimation of high discriminating power is, therefore, possible for patients with localised and metastatic neuroblastoma using stage and MYCN. PMID: 7576963 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 243: Eur J Cancer. 1995;31A(4):520-3. Cytogenetic evolution of MYCN and MDM2 amplification in the neuroblastoma LS tumour and its cell line. Corvi R, Savelyeva L, Amler L, Handgretinger R, Schwab M. Division of Cytogenetics, German Cancer Research Centre, Heidelberg, Germany. Amplification of the MYCN gene is frequently seen either in extrachromosomal double minutes (DMs) or in homogeneously staining regions (HSRs) of aggressively growing neuroblastomas. Total genomic DNA from cell line LS, from early passages of the same line and from original tumour material was biotinylated and hybridised to metaphase chromosomes of normal human lymphocytes. The reverse genomic hybridisation revealed the amplified DNA to be derived both from chromosome 2p23-24, which is the position of MYCN, and from chromosome 12 band q13-14. The MDM2 gene, located at 12q13-14, was found amplified both in early and late passages of LS, in addition to amplified MYCN. Amplification units of MYCN and MDM2 appear first to develop within DMs, which then integrate into different chromosomes to develop to HSRs. PMID: 7576957 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 244: Eur J Cancer. 1995;31A(4):516-9. The mycN/max protein complex in neuroblastoma. Short review. Wenzel A, Schwab M. Department of Cytogenetics, German Cancer Research Center, Heidelberg, Germany. The oncogenic activation by amplification of the MYCN gene is frequently observed in human neuroblastomas and occasionally in other tumours with neuronal qualities. As a consequence of amplification, elevated levels of the mycN protein are expressed. mycN contains a C-terminal basic region (BR) that can bind to DNA, and a helix-loop-helix (HLH)-leucine zipper (Zip) domain, which is responsible for the physical interaction with another HLH-Zip protein, max. This principle structure is conserved among all members of the MYC gene family. The resulting dimers can bind to the DNA sequence CACGTG. The mycN protein, but not max, contains, near the N-terminus, a region conferring the ability to activate the transcription of genes. mycN/max heterodimers probably activate and max/max homodimers repress transcription of, as yet, unidentified target genes. In neuroblastoma cells, where mycN is deregulated, the balanced interaction of BR-HLH-Zip proteins is probably perturbed, and, therefore, genes controlled by mycN might be abnormally expressed and thereby alter normal cell growth with the consequence of tumorigenesis. Publication Types: Review Review, Tutorial PMID: 7576956 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 245: Eur J Cancer. 1995;31A(4):510-5. Regression and progression in neuroblastoma. Does genetics predict tumour behaviour? Ambros PF, Ambros IM, Strehl S, Bauer S, Luegmayr A, Kovar H, Ladenstein R, Fink FM, Horcher E, Printz G, et al. CCRI, St Anna Kinderspital, Vienna, Austria. Neuroblastoma (NB) is a heterogeneous disease. The clinical course may range from spontaneous regression and maturation to very aggressive behaviour. Stage 4s is a unique subcategory of NB, generally associated with good prognosis, despite skin and/or liver involvement and the frequent presence of tumour cells in the bone marrow. Another type of NB is the locally invasive tumour without bone and bone marrow involvement which can also have a good prognosis, irrespective of lymph node involvement. Unfortunately, there is only limited biological information on such tumours which have not been treated with cytotoxic therapy despite lymph node involvement, residual tumour mass after surgery and/or bone marrow infiltration. In order to find specific genetic changes common to NBs with a benign clinical course, we studied the genetic abnormalities of these tumours and compared them with highly aggressive tumours. We analysed a series of 54 localised and stage 4s tumours by means of in situ hybridisation performed on fresh cells or on paraffin embedded tissues. In addition, we performed classical cytogenetics, Southern blotting and PCR analysis on fresh tumour tissue. The majority of patients had been treated with surgery alone, and in a number of patients tumour resection was incomplete. Deletions at 1p36 and amplifications of the MYCN oncogene were absent, and diploidy or tetraploidy were not seen in any case, with residual localised tumours possessing a favourable outcome. Unexpectedly, one patient with a tetraploid 4s tumour without any genetic structural changes not receiving any cytotoxic treatment, did well. Interestingly, this genetic spectrum contrasted with that of progressing tumours, in which most had genetic aberrations, the deletion at 1p36 being the most common event. These data, although limited, suggest that an intact 1p36 (recognised by D1Z2), the absence of MYCN amplification and near-triploidy (at least in localised tumours), represent prerequisites for spontaneous regression and/or maturation. PMID: 7576955 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 246: Eur J Cancer. 1995;31A(4):505-10. Molecular basis for heterogeneity in human neuroblastomas. Brodeur GM. Children's Hospital of Philadelphia, U.S.A. Neuroblastomas demonstrate both clinical and biological heterogeneity. We have proposed that neuroblastomas may be classified in three genetically distinct subtypes, based on cytogenetic and molecular analysis. The first comprises those with hyperdiploid or triploid modal karyotypes (or compatible DNA content by flow cytometry), 1p LOH and MYCN amplification are absent, and TRKA expression is high. These patients are likely to be infants with low stages of disease (stages 1, 2, or 4S by the International Neuroblastoma Staging System), and they have a very favourable outcome (> 90% cure). The second group consists of tumours that generally have a near diploid or tetraploid modal chromosome number or DNA content but lack MYCN amplification. They usually have 1p allelic loss, 14q allelic loss or other structural changes, and TRKA expression is usually low. These patients are generally older with advanced stages of disease (stages 3 or 4), and they have a slowly progressive course, with a cure rate of 25-50%. The third group is characterised by tumours with MYCN amplification. These tumours are generally near diploid or tetraploid, with 1p allelic loss, and low or absent TRKA expression. The patients are usually between 1 and 5 years of age with advanced stages of disease, and they have a very poor prognosis (< 5%). It remains to be determined if tumours in one group ever evolve into a less unfavourable group, but current evidence suggests that they are distinct genetically. The identification of the oncogenes, suppressor genes and growth factor receptor pathways involved in neuroblastomas has provided great insight into the mechanisms of malignant transformation and progression, and ultimately they may provide the targets for future therapy. Publication Types: Review PMID: 7576954 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 247: Eur J Cancer. 1995;31A(4):471-5. CD44 expression and modulation on human neuroblastoma tumours and cell lines. Gross N, Beck D, Beretta C, Jackson D, Perruisseau G. Pediatrics Department, University Hospital, Lausanne, U.K. The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment and tumour metastasis. A large family of variants or isoforms, generated by alternative splicing of a single gene, has been reported to be involved in the malignant process, by conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumour characterised by an aggressive and metastatic behaviour in advanced stages, with amplification of the MYCN protooncogene. In this report, we show that the CD44 standard molecule was highly expressed in the majority of tumours of stages 1-3, in all stage 4s and ganglioneuromas, but only in a subset of stage 4 tumours. A lack of CD44 expression was observed in all MYCN amplified stage 4 tumours, thus demonstrating a highly significant inverse relationship between MYCN amplification and CD44 expression in neuroblastoma. In addition, the expression of 4 different CD44 isoforms was measured on all specimens and was always found to be negative. Using neuroblastoma cell lines and MYCN expressing transfectants, we show that CD44 expression by neuroblastoma cell lines is not directly related to MYCN amplification, but is associated to the stage of differentiation or lineage, and to the tumorigenic properties of the cells. In addition, CD44 expression can be upmodulated parallel to differentiation or maturation as induced by retinoic acid, bromodeoxyuridine or phorbol ester. In contrast, cytokines such as IFN gamma, TNF alpha, or growth factors such as bFGF, SCF and TGF beta were ineffective in modulating CD44 expression. PMID: 7576948 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 248: Int J Cancer. 1994 Oct 1;59(1):141-8. Altered growth and phenotype in clonal mycN transfectants of the SK-N-SH neuroblastoma cell line. Gross N, Miescher G, Beck D, Favre S, Beretta C. Department of Pediatrics, University Hospital, CHUV, Lausanne, Switzerland. We have attempted to distinguish in human neuroblastoma between the effects of mycN on differentiation and its potential to promote malignant progression. Others have observed out-growth of autocrine cells with evidence of an advanced malignant phenotype in a mycN-transfected clonal cell line derived from the single-copy mycN neuroblastoma, SK-N-SH. We have now transfected the parental cell line with the same mycN expression vector and selected 5 clones characterized by unique and stable chromosomal integration sites and variable exogenous copy numbers. mycN gene expression was variable in the different clones and correlated roughly with the copy number of transfected mycN genes. Clones with minimal levels of mycN gene expression had a neuroblastic phenotype and low numbers of surface HLA class-I molecules. Clones with high levels of mycN expression had a Schwann/glial-like phenotype with higher surface HLA class-I display without imbalance of expression of specific loci and accelerated growth. Two such clones were capable of anchorage-independent growth in the absence of serum, and acquired tumorigenic properties. Our results show that exogenous mycN expression can be associated with a differentiation of neuroblastoma cells along the Schwann/glial pathway and can induce accelerated and autonomous growth. PMID: 7927894 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 249: Pediatr Pathol. 1994 Sep-Oct;14(5):823-32. Assessment of MYCN amplification in neuroblastoma biopsies by differential polymerase chain reaction. Boerner S, Squire J, Thorner P, McKenna G, Zielenska M. Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada. Amplification of the oncogene MYCN is a genetic change frequently observed in neuroblastoma and is an indicator of poor prognosis. MYCN copy number is currently determined by Southern blot hybridization. This technique takes 2 to 3 weeks, is labor-intensive, is sensitive to DNA degradation, and requires large quantities of DNA. We have evaluated a new, semiquantitative method of estimating gene copy number that uses differential polymerase chain reaction (PCR). The procedure can be performed in 1 day, is highly reproducible, and requires only nanogram quantities of DNA. It employs a semiquantitative, nonisotopic PCR technique based on differential competition for PCR substrates. MYCN gene primers are amplified together with primers from a single-copy internal control gene. Following electrophoretic separation, the ratio of the two PCR products is determined visually and by densitometric analysis of ethidium bromide-stained agarose gels. This differential ratio is then compared to a series of ratios generated from standards of known MYCN gene copy number. We compared the results obtained by this differential PCR method with those obtained by conventional Southern blotting in 16 cases of primary neuroblastoma. All amplified tumors were detected by differential PCR, and no false positives were observed. We confirmed that differential PCR is a rapid and reliable alternative to Southern blotting for MYCN copy number assessment and is highly suited to the analysis of DNA derived from needle biopsies. PMID: 7808981 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 250: Cancer Res. 1994 Aug 1;54(15):4238-42. CD44H expression by human neuroblastoma cells: relation to MYCN amplification and lineage differentiation. Gross N, Beretta C, Peruisseau G, Jackson D, Simmons D, Beck D. Pediatrics Department, University Hospital, Lausanne, Switzerland. The human CD44 cell surface glycoprotein has been involved in a variety of functions including lymphocyte homing, extracellular cell matrix attachment, and tumor metastasis. Due to the alternative splicing of the single gene, a large family of different variants or isoforms is generated. Several reports have indicated an up-regulation of CD44 variant (v) isoforms in malignant process, conferring metastatic potential to non-metastatic cells. Neuroblastoma is a tumor characterized by an aggressive and metastatic behavior in advanced stages with amplification of the MYCN protooncogene. In this report we show that the CD44 standard molecule is highly expressed in 100% of stage I-III, IVs neuroblastomas and ganglioneuromas but only in a subset of stage IV tumors. In contrast, no expression of CD44 was detected on MYCN amplified stage IV tumors, thus demonstrating a highly significant negative relationship between MYCN amplification and CD44 expression in neuroblastoma. The expression of CD44 on neuroblastoma cultured cell lines was not shown to be related to MYCN amplification but rather linked to the S-type, schwann/glial differentiation lineage. Immunochemical analysis of tumor samples with anti-CD44v3 and -v6 antibodies and Northern blot analysis of mRNA from cell lines with probes spanning exons 4-10 did not reveal any expression of splice variants on neuroblastomas of all stages and cell lines, thus ruling out a major role of these isoforms in neuroblastoma progression and metastasis. PMID: 7518353 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 251: Genes Chromosomes Cancer. 1994 Jul;10(3):213-6. Characterization of homogeneously staining regions in a small cell lung cancer cell line, using in situ hybridization with an MYCN probe. Dietzsch E, Lukeis RE, Vrazas V, Hasthorpe S, Garson OM. University of Melbourne Department of Medicine, Australia. The cell line CIPL38 was derived from the pleural effusion of a patient with small cell lung cancer. The karyotype was hyperdiploid and complex with a variable number of marker chromosomes. Two of the markers had large homogeneously staining regions (hsr), which were shown to consist of amplified MYCN by in situ hybridization. One hsr bearing a marker chromosome could not be identified with G-banding, but the other was situated on a der(14). This was elucidated further with FISH analysis, which enabled the identification of sequences of chromosome i involved in a complex rearrangement with chromosome 14 and the hsr. PMID: 7522047 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 252: Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5523-7. MYCN is retained in single copy at chromosome 2 band p23-24 during amplification in human neuroblastoma cells. Corvi R, Amler LC, Savelyeva L, Gehring M, Schwab M. Department of Cytogenetics, German Cancer Research Center, Heidelberg. Amplification of the human N-myc protooncogene, MYCN, is frequently seen either in extrachromosomal double minutes or in homogeneously staining regions of aggressively growing neuroblastomas. MYCN maps to chromosome 2 band p23-24, but homogeneously staining regions have never been observed at this band, suggesting transposition of MYCN during amplification. We have employed fluorescence in situ hybridization to determine the status of MYCN at 2p23-24 in five human neuroblastoma cell lines. All five lines carried, in addition to amplified MYCN in homogeneously staining regions or double minutes, single-copy MYCN at the normal position. In one line there was coamplification of MYCN together with DNA of the host chromosome 12, to which MYCN had been transposed. Our results suggest a model of amplification where MYCN is retained at its original location. They further sustain the view that either the initial events of MYCN amplification or the further evolution of amplified MYCN copies follow mechanisms different from those leading to amplification of drug-resistance genes. PMID: 8202521 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 253: Pediatr Med Chir. 1994 May-Jun;16(3):211-8. [The prognostic effect of amplification of the MYCN oncogene in neuroblastoma. The preliminary results of the Italian Cooperative Group for Neuroblastoma (GCINB)] [Article in Italian] Pession A, De Bernardi B, Perri P, Mazzocco K, Rondelli R, Nigro M, Iolascon A, Forni M, Basso G, Conte M, et al. Dipartimento di Biologia, Universita di Bologna, Italia. Of 567 children with neuroblastoma diagnosed between November 1984 and May 1993 in 21 Italian institutions, 235 (41%) have been evaluated for MYCN oncogene amplification. The amplification (3 or more copies of the gene) was found in 39 patients (17%) and was more frequent in patients aged more than one year, abdominal primary site of the tumor, advanced stages, normal urinary excretion of vanillylmandelic acid (VMA), and high level of LDH, NSE and ferritin. The five-year survival of the 235 patients (62%) was significantly better in patients with normal copy number of MYCN (69% versus 29%). By correlating genomic amplification with clinical and biochemical characteristics, MYCN amplification was found associated with a worse prognosis even when patients were subdivided for age (under and above one year), disease extension (localized operable, localized but inoperable, and disseminated) with exception for Stage IV-S, VMA and homovanillic acid excretion, serum levels of NSE and ferritin, but not of LDH. These data confirm the unfavourable prognostic meaning of MYCN amplification, but are unable to define if it represents a new independent variable. Publication Types: Multicenter Study PMID: 7971442 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 254: Genes Chromosomes Cancer. 1994 May;10(1):30-9. There may be two tumor suppressor genes on chromosome arm 1p closely associated with biologically distinct subtypes of neuroblastoma. Takeda O, Homma C, Maseki N, Sakurai M, Kanda N, Schwab M, Nakamura Y, Kaneko Y. Department of Laboratory Medicine, Saitama Cancer Center Hospital, Japan. We studied loss of heterozygosity (LOH) on chromosome arm 1p in 108 neuroblastomas using 14 polymorphic DNA markers. One-hundred and four tumors with one or more informative loci; 21 (20%) of the 104 tumors showed LOH on 1p, and were classified into three groups on the basis of interstitial or terminal allelic loss, and presence or absence of LOH on 1p. Seven of the 21 tumors showed an interstitial deletion which encompassed a small region in 1p36 (group A), and the other 14 showed a terminal deletion which encompassed the region from 1pter to 1p32 (group B). Eighty-three tumors without LOH on 1p were classified as group C. The group A patients were mostly less than 12 months of age (6/7), were frequently found by a mass screening program for infants (5/7), had a tumor of non-adrenal origin, and rarely progressed to stage IV (1/7). Most group B patients were 12 months or older (11/14), were found clinically (11/14), had tumors of adrenal origin, and progressed to stage IV (10/14). Analysis of biologic characteristics in group C tumors suggested that they may comprise group A and B tumors. While all group A tumors were in the triploid range (3n) (4/4), most group B tumors were diploid (2n) or tetraploid (4n) (7/10). MYCN amplification was found in 8 group B tumors, but in none of group A tumors. Event-free survivals of groups A, B, and C patients at 3 years were 86, 49, and 74%, respectively (P = 0.0287). These findings suggest that there may be two tumor suppressor genes on 1p which are closely associated with two biologically distinct subtypes of neuroblastoma. PMID: 7519871 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 255: Cancer Res. 1994 Apr 15;54(8):2256-61. Coactivation of the MDR1 and MYCN genes in human neuroblastoma cells during the metastatic process in the nude mouse. Ferrandis E, Da Silva J, Riou G, Benard I. Laboratory of Clinical and Molecular Pharmacology, Institut Gustave Roussy, Villejuif, France. In metastatic human neuroblastoma, MYCN amplification and MDR1 overexpression are frequently observed. No in vivo model is yet available for the study of the regulation of these two genes during the metastatic process of this disease. Culture of an involved bone marrow of a patient with stage IV neuroblastoma gave rise to an established in vitro neuroblastoma cell line, IGR-N-91, and a subsequent s.c. xenograft model in nude mice. When cultured in vitro, blood cells, bone marrow, and the myocardium of mice bearing s.c. tumor xenograft reproducibly yielded cells with morphological and molecular features of neuroblastoma cells, including consistent MYCN amplification (60 copies/haploid genome). Compared to the neuroblastoma cells of the primitive s.c. tumor xenograft, metastatic cells showed a significant increase in the MYCN gene transcript levels associated with an overexpression of the MDR1 gene mRNA levels leading to a P-glycoprotein capable of extruding Adriamycin. This study offers compelling evidence that (a) IGR-N-91 is a human neuroblastoma xenograft model able to induce metastasis in nude mice, (b) an increase in MYCN and MDR1 transcripts levels is associated with the metastatic process, and (c) IGR-N-91 provides a biological tool for the study of gene activations during tumor dissemination in neuroblastoma. PMID: 8174136 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 256: Cancer. 1994 Apr 15;73(8):2231-7. MYCN gene amplification in rhabdomyosarcoma. Driman D, Thorner PS, Greenberg ML, Chilton-MacNeill S, Squire J. Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada. BACKGROUND. Amplification of the MYCN oncogene, formerly known as N-myc, has been seen in several malignant tumors, particularly neuroblastoma, where its association with a poor clinical outcome is the clearest example of a clinically relevant oncogene mutation in any human cancer. METHODS. The incidence and clinical significance of MYCN amplification in rhabdomyosarcoma (RMS) was assessed by Southern blot analysis in this retrospective study of seven alveolar RMS and six embryonal RMS. RESULTS. MYCN amplification (4- to 13-fold) was present in three of seven alveolar RMS (42.9%) but in none of the embryonal RMS. There was no significant difference between the clinical behavior of the MYCN-amplified and unamplified tumors, and no correlation was found with the light microscopic appearances of the tumors or with desmin immunoreactivity. CONCLUSIONS. The findings are compatible with previous studies that demonstrated cytogenetic evidence of gene amplification in RMS, and help to clarify conflicting reports in the literature about MYCN amplification in alveolar and embryonal RMS. The results raise the possibility of important biologic differences between these subtypes of RMS, differences that warrant further investigation. PMID: 8156531 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 257: Cancer. 1994 Apr 1;73(7):1987-93. Pediatric malignant glioma with tubuloreticular inclusions and MYCN amplification. Report of a case with immunohistochemical, ultrastructural, flow cytometric, karyotypic, and Southern blot analysis. Jay V, Rutka J, Becker LE, Squire J. Department of Pathology, Hospital for Sick Children-University of Toronto, Ontario, Canada. BACKGROUND. The authors described unusual pathologic features in a left frontal lobe malignant glioma in a 31/2-year-old boy. The pathology was similar in the initial excision and two subsequent recurrences at 9 and 11 months and at autopsy, when extensive subarachnoid spread was noted. METHODS. The tumor was studied by conventional histology, immunohistochemistry, flow cytometry, transmission electron microscopy (TEM), immune electron microscopy (IEM), and cytogenetic and Southern blot analysis. RESULTS. The tumor revealed two different histologic patterns. One component showed large cells with eosinophilic cytoplasm, vesicular nuclei with prominent nucleoli, eosinophilic perinuclear inclusions, and immunoreactivity for glial fibrillary acidic protein (GFAP) and vimentin. The other component consisted of undifferentiated cells with hyperchromatic nuclei and scanty cytoplasm. By TEM, the perinuclear aggregates were composed of tubuloreticular inclusions, which were also observed in endothelial cells within the tumor vasculature. By IEM, the intermediate filaments in the tumor cell cytoplasm were decorated with GFAP. Flow cytometric results revealed a marked increase in the S-phase (48%), whereas cytogenetic analysis of short-term cultures showed an abnormal karyotype containing marker chromosomes and double minutes. In the second resection, additional karyotypic abnormalities were noted, including 1p- and several additional markers. The first and second resections showed MYCN amplification by Southern Blot analysis in the 60- to 80-fold range. CONCLUSIONS. This tumor presents unique histologic, ultrastructural, and cytogenetic findings as well as MYCN amplification that is notable for a pediatric malignant glioma. Tubuloreticular inclusions were a prominent feature in this tumor, which again is unique for a glioma. Publication Types: Case Reports PMID: 8137227 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 258: J Clin Endocrinol Metab. 1994 Feb;78(2):387-92. Molecular genetic studies of sporadic pituitary tumors. Boggild MD, Jenkinson S, Pistorello M, Boscaro M, Scanarini M, McTernan P, Perrett CW, Thakker RV, Clayton RN. Department of Medicine, Keele University, Stoke-on-Trent, United Kingdom. Tumor formation may result from the activation of dominant oncogenes or by inactivation of recessive, tumor suppressor genes. The role of such mutations in the development of pituitary tumors has been studied. Tumors from 88 patients, representing the 4 major classes of adenoma, were investigated. In DNA extracted from matched leukocyte and tumor samples, allelic deletions were sought with 15 probes identifying restriction fragment length polymorphisms on chromosomes 1, 5, 10, 11, 13, 17, 20, and 22. Evidence of amplification or rearrangement of 10 recognized cellular oncogenes (N-ras, mycL1, mycN, myc, H-ras, bcl1, H-stf1, sea, kraS2, and fos) was sought in tumor DNA. Activating dominant mutations of Gs alpha were detected using the polymerase chain reaction to amplify exons 7-10 and hybridizing the product to normal and mutant allele-specific oligonucleotides. Allelic deletions on chromosome 11 were identified in 16 tumors (18%) representing all 4 major subtypes. Deletions on other autosomes were observed in less than 6% of tumors. Three adenomas had deletions on multiple autosomes, 2 of these were aggressive and recurrent. Mutations of Gs alpha were confirmed to be specific to somatotrophinomas, being identified in 36% of such tumors in this series. No evidence of amplification or rearrangement of other recognized cellular oncogenes was found. Inactivation of a recessive oncogene on chromosome 11 is an important and possibly early event in the development of the four major types of pituitary adenoma, whereas activating mutations of Gs alpha are confirmed to be specific to somatotropinomas. Two aggressive tumors were found to have multiple autosomal losses, suggesting a multistep progression in the development of tumors of this phenotype. PMID: 8106627 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 259: Genes Chromosomes Cancer. 1994 Feb;9(2):129-35. Enhanced MYCN expression and isochromosome 17q in pineoblastoma cell lines. Kees UR, Biegel JA, Ford J, Ranford PR, Peroni SE, Hallam LA, Parmiter AH, Willoughby ML, Spagnolo D. Division of Children's Leukemia and Cancer Research, Western Australian Research Institute for Child Health, Princess Margaret Hospital, Perth. We have established two cell lines, PER-452 and PER-453, from an 8-month-old girl with an extensive pineoblastoma. Characterization of these lines revealed that the proto-oncogenes MYC and MYCN were not amplified, but both cell lines showed MYCN expression comparable to a cell line with 200-fold MYCN amplification. Both cell lines contained an i(17q). These results support the concept that pineoblastomas belong to a larger group of primitive neuroectodermal tumors of the central nervous system. These two cell lines provide a unique opportunity to investigate the molecular genetic mechanisms underlying these neoplasms further. Publication Types: Case Reports PMID: 7513543 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 260: Cytogenet Cell Genet. 1994;66(1):75-6. High resolution mapping of the MYCN proto-oncogene at human chromosome 2p24.3 by fluorescence in situ hybridisation. Shibasaki Y. MRC Human Genetics Unit, Edinburgh, Scotland, UK. The MYCN proto-oncogene was previously mapped to human chromosome 2p24.1 by analysis of mouse x human somatic cell hybrids and radioactive in situ hybridisation to normal human chromosomes. However, using fluorescence in situ hybridisation (FISH) and high resolution chromosome banding techniques MYCN has been reassigned to 2p24.3. PMID: 8275716 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 261: Prog Clin Biol Res. 1994;385:111-6. Prognostic value of MDR1 gene expression in neuroblastoma: results of a multivariate analysis. Benard J, Bourhis J, de Vathaire F, Ferrandis E, Terrier-Lacombe MJ, Lemerle J, Riou G, Hartmann O. Laboratory of Molecular and Clinical Pharmacology, Institut Gustave Roussy, Villejuif, France. The prognostic value of the MDR1 gene expression in neuroblastoma (NB) was assessed in a multivariate analysis performed in a series of 84 patients (pts) taking into account the main known clinical and biological factors of the disease, i.e., age, stage, MYCN genomic content and DNA ploidy index. Twenty seven children were < 1 year (yr), 13 presented with stage I and II, 7 with stage IV-S, 17 with stage III and 47 (56%) with stage IV. Tumor specimens were obtained from involved bone marrow (n = 12) or surgical primary tumor specimens (n = 72). MDR1 gene expression was measured by Northern hybridization technique and expressed in arbitrary units (a. u.) (Goldstein et al., 1989). Analysis of MYCN genomic content and DNA ploidy index were performed by Southern blot hybridization technique and flow cytometry, respectively. Out of 84 tumor specimens 19 (23%) showed MYCN amplification (> 3 copies/haploid genome). In 24 cases (29%) no detectable MDR1 gene transcript was found (0 a.u.) whereas 42 (50%) had a value in the range 1-30 a.u., and 18 (21%) a value beyond 30 a.u.. High transcript levels were found in localized as well as in metastatic NB (NS). No significant correlation between MDR1 gene expression, age, stage, or MYCN genomic content was found In univariate analysis stage IV, age > 1 yr, MYCN amplification, diploid DNA content and high MDR1 gene transcript levels were significantly related to an increased risk of death. In multivariate analysis only stage IV, MYCN amplification and MDR1 overexpression remained significantly associated with an increased risk of death.(ABSTRACT TRUNCATED AT 250 WORDS) PMID: 7972202 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 262: Genes Chromosomes Cancer. 1993 Sep;8(1):15-21. Structural organization of MYCN amplicons of neuroblastoma tumors, xenografts, and cell lines characterized by the sequences encompassing the MYCN amplicons in a human neuroblastoma cell line. Akiyama K, Kanda N, Yamada M, Kato M, Tadokoro K, Matsunaga T, Nishi Y. Life Science Research Laboratory, Japan Tobacco, Inc., Kanagawa. We characterized differences in the structural organization of the MYCN amplicons of a number of neuroblastomas by analyzing 8 contigs spanning 330 kb cloned from the MYCN amplicon of a neuroblastoma cell line. Some regions were amplified in almost all specimens, the conserved regions (CRs), and others were differentially amplified in some subsets, the non-conserved regions (NCRs). CRs constituted only 20% of the 330 kb region, with the remainder being NCRs. The regions that inevitably co-segregate with the MYCN gene make up the core, whereas flanking regions are retained at random. If a histogram of the frequency with which the amplified NCR sequences from one specimen match those of the cell line MC-NB-I shows a random distribution, the NCRs would co-segregate with MYCN as a result of random events. However, both the tumors and cell lines/xenografts showed a distribution with two distinct peaks; one from a group containing a small number of sequences with a fairly high degree of homology to the NCRs of MC-NB-I, and the other from a group containing a large number of sequences with little homology. These results indicate that the flanking segments are preferentially co-segregated with MYCN by a non-random mechanism. PMID: 7691154 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 263: Lab Invest. 1993 Jul;69(1):43-50. Detection of MYCN gene amplification and deletions of chromosome 1p in neuroblastoma by in situ hybridization using routine histologic sections. Leong PK, Thorner P, Yeger H, Ng K, Zhang Z, Squire J. Department of Pathology, Hospital for Sick Children, Toronto, Canada. BACKGROUND: Amplification of the MYCN oncogene and partial deletion of chromosome 1p are genetic changes frequently seen in neuroblastoma that are indicators of poor prognosis. The identification techniques usually used--Southern blotting for MYCN gene amplification, and karyotypic analysis of viable tumor cells for large 1p deletions--are time-consuming and suited for specialized laboratories. EXPERIMENTAL DESIGN: We have developed an immunofluorescence in situ hybridization assay suitable for detection of MYCN amplification and 1p deletion in formalin-fixed, paraffin-embedded tissue sections. The technique is rapid, does not involve the use of radioactivity, and can be carried out in laboratories already familiar with conventional in situ hybridization and immunohistochemical detection methods. RESULTS: MYCN gene amplification appeared as multiple signals per nucleus, corresponding to double minute chromosomes. Deletion of 1p was detected by loss of one of the normal signals present within the nucleus of a neuroblastoma cell line. Use of different fluorophores enabled the simultaneous detection of MYCN gene amplification and 1p deletion at the individual cell level. Detection was found to be enhanced by RNase and pepsin treatment of tissue sections before hybridization and by a novel microwave denaturation method. CONCLUSIONS: Application of this methodology to formalin-fixed samples of neuroblastoma will permit comprehensive retrospective studies of these two genetic markers using archived tumors. In situ hybridization surveys will have widespread applications for studying known genetic aberrations in individual cells of a variety of solid tumors and for determining interrelationships and clinical significance in relation to tumor progression and patient outcome. PMID: 8331897 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 264: J Natl Cancer Inst. 1993 Mar 3;85(5):377-84. Comment in: J Natl Cancer Inst. 1993 Mar 3;85(5):344-5. Lack of high-affinity nerve growth factor receptors in aggressive neuroblastomas. Suzuki T, Bogenmann E, Shimada H, Stram D, Seeger RC. Department of Pediatrics, Childrens Hospital Los Angeles, University of Southern California School of Medicine 90054-0700. BACKGROUND: Neuroblastoma is a malignancy of the sympathetic nervous system. Nerve growth factor, which has a major role in development of the sympathetic nervous system, has high-affinity (gp140TRK-A) and low-affinity (gp75NGFR) cell-surface receptors. We recently reported preliminary study results showing a lack of gp140TRK-A receptors and rapid disease progression in neuroblastomas, particularly those with amplification of the N-myc (also known as MYCN) proto-oncogene. PURPOSE: This retrospective study was designed to determine if expression of nerve growth factor receptor messenger RNA (mRNA) was associated with biologic and clinical parameters and with survival in neuroblastoma. METHODS: We obtained 80 untreated primary neuroblastomas that had been snap-frozen and stored after surgical excision. To determine expression of gp140TRK-A and gp75NGFR, we performed Northern blot analyses on total RNA from the specimens. Samples from the same specimens were examined for N-myc proto-oncogene amplification, RNA expression, and histologic differentiation, and clinical stage at diagnosis and survival were determined. RESULTS: Of the 80 neuroblastomas, 65 (81%) expressed gp140TRK-A RNA. However, three (27%) of the 11 tumors with genomic amplification and high expression of N-myc RNA and 62 (90%) of the 69 without genomic amplification or detectable N-myc RNA expressed gp140TRK-A mRNA. The inverse relationship between gp140TRK-A mRNA and N-myc expression had high statistical significance (P < .0001). Of the 67 tumors assessable for histologic differentiation, the 13 lacking gp140TRK-A mRNA were histologically undifferentiated, whereas 19 (35%) of the 54 expressing it were differentiated (P = .041). Only 10 (53%) of the 19 metastatic (stage IV) tumors expressed gp140TRK-A mRNA, compared with 90% for other stages (P = .0003). Survival 2 years after diagnosis was 92%, 78%, and 14% for patients whose tumors expressed high, intermediate, and no gp140TRK-A mRNA, respectively (P < .0001). Univariate and multivariate analyses demonstrated that N-myc and gp140TRK-A expression of mRNA and clinical staging were independent predictors of survival. Expression of gp75NGFR mRNA did not correlate with gp140TRK-A mRNA expression, histologic differentiation, stage, or survival. CONCLUSIONS: The expression of gp140TRK-A mRNA correlates with distinct biologic and clinical subsets of neuroblastoma, which suggests a role for the high-affinity nerve growth factor receptors in determining the phenotype of neuroblastoma. The absence of gp140TRK-A mRNA expression, whether or not the N-myc proto-oncogene is amplified, is associated with tumor progression. PMID: 8433391 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 265: Eur J Cancer. 1993;29A(6):802-4. Neuroblastoma: a multiple biological disease. Tonini GP. Department of Hematology and Oncology, G. Gaslini Children's Hospital, Genova, Italy. Neuroblastoma (NB) is a paediatric tumour showing an appreciable variability in clinical evolution. Localised tumours (especially stage 1) can be mildly treated with good success while metastatic tumours (stage 4) are highly aggressive. This suggests a great biological diversity. In fact, molecular and genetic studies have revealed distinct abnormalities in localised and non-localised tumours. Loss of heterozygosity for the short arm of chromosome 1, 1p deletion, and MYCN amplification are present in stages 3 and 4 but rarely in stages 1 and 2. Metastatic stage 4S in infants is peculiar and does not show the same genetic and molecular abnormalities found in advanced metastatic tumours. Considering the biological alterations associated with NB, it would appear that advanced stage NB conforms to the multistep model of tumour development while stage 4S can be divided into two groups: one arising from a lack of cellular differentiation and the other as a consequence of an additional 'one hit' mutation. PMID: 8484968 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 266: Pediatr Hematol Oncol. 1993 Jan-Mar;10(1):31-4. MYCN amplification by differential PCR. Huddart SN, Mann JR, McGukin AG, Corbett R. Department of Oncology, Children's Hospital, Birmingham, United Kingdom. A method is described to estimate MYCN (N-myc) oncogene amplification in neuroblastoma by the technique of differential polymerase chain reaction (PCR). The technique is quicker than conventional Southern blotting techniques and does not require radioactive materials. The ability to measure MYCN amplification from smaller amounts of tumor DNA also permits measurement from Tru-cut biopsy samples and opens the possibility of retrospective measurement of MYCN status from single paraffin sections of archival material. PMID: 8443050 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 267: Diagn Mol Pathol. 1992 Dec;1(4):229-34. Rapid detection of MYCN gene amplification in neuroblastomas using the polymerase chain reaction. Crabbe DC, Peters J, Seeger RC. Department of Pediatrics, Children's Hospital Los Angeles, CA 90054-0700. We have used the polymerase chain reaction (PCR) to detect amplification of the MYCN oncogene in neuroblastoma cell lines and to distinguish primary tumors with a single copy from those with MYCN amplification using DNA extracted from frozen sections. Two primer pairs were used to co-amplify a 428-bp fragment of the MYCN oncogene along with a 268-bp fragment of the beta-globin gene (a single-copy reference standard). After 30 cycles of PCR, the products were resolved by agarose gel electrophoresis. MYCN gene amplification was identified by visual comparison of the relative intensities of MYCN and beta-globin PCR product bands on the ethidium bromide-stained gel. This semiquantitative approach, while inadequate for precise determination of copy number, provided a simple, rapid, nonisotopic method for differentiating tumors with MYCN amplification from those with a single copy. Seventy-four primary tumors were classified as amplified or nonamplified by semiquantitative PCR. Twenty-two of 23 tumors known to carry MYCN gene amplification by Southern analysis were correctly identified by PCR. The single false-negative result was due to a sampling error: DNA was extracted from a block of tissue containing small foci of tumor surrounded by normal tissue. Fifty-one of 51 tumors with a single copy of MYCN were also correctly identified by PCR. We conclude that semiquantitative PCR is a reliable, non-isotopic alternative to Southern blotting for detection of MYCN gene amplification that can be performed rapidly on DNA extracted from frozen sections. PMID: 1342970 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 268: Cancer. 1992 Nov 15;70(10):2444-50. Molecular genetic, cytogenetic, and immunohistochemical characterization of alveolar soft-part sarcoma. Implications for cell of origin. Cullinane C, Thorner PS, Greenberg ML, Kwan Y, Kumar M, Squire J. Department of Pathology, Hospital for Sick Children, Toronto, Ontario, Canada. BACKGROUND. Alveolar soft-part sarcoma is a rare tumor of uncertain histogenesis. METHODS. The authors report a patient who was studied using immunohistochemistry, cytogenetic analysis, and molecular probes for MyoD1 and MYCN (N-myc proto-oncogene). RESULTS. By immunoperoxidase, the tumor was focally positive for vimentin, neuron-specific enolase, and S-100 protein but negative for muscle-specific actin, desmin, and low-molecular-weight keratin. Direct chromosome analysis of primary tumor cells using G-banded preparations yielded two clonally abnormal lines: one demonstrated trisomy 47,XX+5; the other demonstrated 46,XX,1p-,17q+. Expression of the MYCN RNA was detectable at a low level, and MYCN was single copy at the DNA level. Expression of the myogenic molecular marker MyoD1 was not detected by Northern blotting analysis. CONCLUSIONS. This is the first detailed study to address the molecular biology and tumor cytogenetics of alveolar soft-part sarcoma. The results of this study indicate a neurogenic origin for this unusual tumor and fail to provide support for the notion of a myogenic origin. Publication Types: Case Reports PMID: 1423174 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 269: Genes Chromosomes Cancer. 1992 Jun;4(4):314-20. Detection of amplified DNA sequences in human tumor cell lines by fluorescence in situ hybridization. Bar-Am I, Mor O, Yeger H, Shiloh Y, Avivi L. Department of Human Genetics, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel. An unambiguous and rapid characterization of amplified DNA sequences in tumor cells is important for the understanding of neoplastic progression. This study was conducted to evaluate the potential of fluorescence in situ hybridization (FISH) to identify such amplified DNA sequences in human tumor cell lines. Applying this technique, we followed the metaphase location and interphase position of amplified DNA sequences corresponding to the SAMK, MYC, and MYCN genes in four cell lines derived from human tumors: two gastric carcinoma lines (KATO III and SNU-16), a neuroblastoma (NUB-7), and a neuroepithelioma (NUB-20) line. In metaphase cells of KATO III, NUB-7, and NUB-20 lines, the amplified regions were clearly visible and easily identified at an intrachromosomal location: in KATO III and NUB-7 at a terminal position and in NUB-20 at an interstitial position. In SNU-16, on the other hand, the amplified SAMK and MYC sequences were identified in extrachromosomal double minute chromosomes (DMs). In this line, the SAMK and MYC sequences were coamplified in the same cells and were colocated on the same DMs. FISH also allowed the identification of amplified DNA sequences in nondividing cells, enabling us to distinguish, at interphase, whether the amplification gave rise to intrachromosomal amplified regions (IARs) or to extrachromosomal DMs. The FISH technique also allowed us to determine at metaphase as well as at interphase the extent of amplification and the size of the IARs. PMID: 1377938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 270: Int J Cancer. 1992 May 8;51(2):250-8. Bi-modal differentiation pattern in a new human neuroblastoma cell line in vitro. Matsushima H, Bogenmann E. Department of Pediatrics, Jikei University School of Medicine, Tokyo, Japan. We have isolated a human neuroblastoma (NB) cell line, HTLA230, from the bone-marrow aspirate of a patient with stage-IV disease. Subcutaneous tumors after inoculation of HTLA230 cells into nude mice were composed of primitive neuroblasts which rarely contained neuro-secretory granules. Cytogenetic studies of the cell line demonstrated 2 distinct populations of cells with common chromosomal markers. Stable sub-clones with a differentiated or undifferentiated cell morphology were isolated, demonstrating phenotypical heterogeneity of the HTLA230 parental cell line. Treatment with retinoic acid (RA) induced extensive neurite outgrowth in the parental cell line and in phenotypically differentiated sub-clones, but rarely in undifferentiated ones. Long-term treatment with RA was not associated with down-modulation of mycN-gene expression, which could be achieved only in cultures treated additionally with aphidicolin, a DNA-synthesis inhibitor, thus eliminating growing NB cells. A RA resistant subclone (CI-5) was isolated from parental HTLA230 cells grown at clonal cell density. Cells originally showed a homogeneously differentiated morphology; however, flat cells (F-cells) appeared with time and were subsequently separately propagated. Transdifferentiation of isolated F-cells into cells with neuron-like (N-cell) morphology was observed. Immunohistochemical analysis demonstrated that F-cells had lost the expression of neuronal markers, including HNK-I and A2B5, and expressed the intermediate filament, vimentin. Furthermore, F-cells showed high incorporation of [methyl-3H] thymidine (3H-TdR) by autoradiography but no mycN protein could be detected, although present in the parental cell line. These results then suggest that the isolated NB cell line and the RA-resistant variant line represent an excellent in vitro model with which the bi-modal differentiation pathway of NB can be analyzed on a molecular biological level. PMID: 1568793 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 271: Cancer Genet Cytogenet. 1992 Apr;59(2):128-34. Detection of MYCN amplification in three neuroblastoma cell lines by non-radioactive chromosomal in situ hybridization. McRobert TL, Rudduck C, Kees UR, Garson OM. Department of Cytogenetics, St. Vincent's Hospital, Fitzroy, Victoria, Australia. A non-radioactive chromosomal in situ hybridization technique utilizing a biotin-streptavidin-polyalkaline-phosphatase complex was successfully applied to three neuroblastoma cell lines for detection of MYCN amplification. These cell lines, designated PER-106, PER-107, and PER-108, were derived from consecutive bone marrow samples taken from a patient with stage IV neuroblastoma. The cell line derived at diagnosis (PER-106) exhibited MYCN amplification in the form of variable numbers of double-minute chromosomes, small fragments, and rings of varying sizes. This observed variability of MYCN amplification may explain the reported heterogeneity of both MYCN mRNA and protein expression among individual cells of some neuroblastomas. The cell lines derived from subsequent samples (PER-107 and PER-108) contained amplified MYCN as two consistent homogeneously staining regions in every cell. These were located on the short arms of chromosomes 6 and 14. Thus, amplified MYCN was identified in each cell line and demonstrated the concurrent evolution of amplification with cytogenetic abnormalities. PMID: 1581879 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 272: Cancer Genet Cytogenet. 1992 Apr;59(2):119-27. Three neuroblastoma cell lines established from consecutive samples of one patient which show distinct morphologic features, MYCN amplification, and surface marker expression. Kees UR, Ford J, Dawson VM, Ranford PR, Armstrong JA. Children's Leukaemia and Cancer Research Unit, Western Australia Research Institute for Child Health, Princess Margaret Hospital, Perth. Three neuroblastoma cell lines established from tumor samples obtained from one patient are described. The three lines were derived from bone marrow aspirates taken at diagnosis, and 13 and 15 months later. The origin of the cell lines PER-106, PER-107, and PER-108 from malignant neuroblasts was confirmed by electron microscopy studies, surface marker analysis, and assessment of MYCN amplification. Cell lines PER-107 and PER-108, which were established from tumor samples obtained at the time of progressive disease, have significantly shorter doubling times than PER-106 and grow mainly substrate-adherent, while cell line PER-106 (established from sample obtained at diagnosis) consists of small round neuroblastic cells which form large aggregates in suspension culture. The electron microscopy studies revealed distinctive neuroblast-like ultrastructure in all cell lines. The MYCN copy number was amplified (greater than 10 copies) in the established cell lines and in the fresh tumor samples and the relative abundance of MYCN RNA in the cell lines correlated roughly with the extent of the MYCN gene amplification. However, distinct phenotypic differences can be demonstrated among these three lines, which provide a model for the further examination of this highly malignant tumor. Detection of MYCN amplification by chromosomal in situ hybridization was performed on this set of cell lines, as reported by McRobert et al. (this issue, pages 128-134). PMID: 1581878 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 273: Genes Chromosomes Cancer. 1992 Apr;4(3):195-204. Two malignant peripheral primitive neuroepithelial tumor cell lines established from consecutive samples of one patient: characterization and cytogenetic analysis. Kees UR, Rudduck C, Ford J, Spagnolo D, Papadimitriou J, Willoughby ML, Garson OM. Children's Leukaemia and Cancer Research Unit, Western Australian Research Institute for Child Health, Princess Margaret Hospital, Perth. A 6-year-old girl presented with a tumor of the right shoulder involving bone, adjacent soft tissue, and regional lymph nodes. The conventional histologic diagnosis was ambiguous, initially suggesting lymphoma. After relapse on lymphoma therapy, reevaluation with additional multiple diagnostic techniques performed on the biopsy tissue and on two cell lines derived from the biopsies established the diagnosis of a primitive neuroepithelial tumor of bone and soft tissue. This was strongly supported by 1) focal rosette formation by the tumor cells and positive immunostaining for neuron-specific enolase and synaptophysin, with absent staining for leukocyte common antigen; 2) at the ultrastructural level, formation of cellular processes containing microtubules, a paucity of neurosecretory granules, absence of synaptic junctions, formation of long "intermediate" junctions between cells, and, in culture, widespread development of rosettes; 3) marked surface positivity to W 6/32 and negativity to HSAN 1.2 antibodies; and 4) elevated expression of MYC and lack of overexpression of MYCN oncogenes. Numerical and structural abnormalities were present in the karyotype, but the expected t(11;22)(q24;q12) was not present in the tumor-involved marrow or in either of the established tumor cell lines, although there was an interstitial deletion of 11q involving breakpoints in q21 and q23. Publication Types: Case Reports PMID: 1382559 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 274: Nucleic Acids Res. 1992 Mar 11;20(5):1165. Tetranucleotide repeat polymorphism at the human N-MYC gene (MYCN). Fougerousse F, Meloni R, Roudaut C, Beckmann JS. Centre d'Etude du Polymorphisme Humain, Paris, France. PMID: 1549499 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 275: Oncogene. 1991 Oct;6(10):1745-51. The MYCN protein of human neuroblastoma cells is phosphorylated by casein kinase II in the central region and at serine 367. Hamann U, Wenzel A, Frank R, Schwab M. Institut fur Experimentelle Pathologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany. The MYCN gene has been implicated in certain neuronal tumours, such as neuroblastomas and retinoblastomas. These tumours express high levels of mRNA and protein of MYCN as a result of amplification. MYCN encodes a short-lived nuclear phosphoprotein whose function has not yet been elucidated. This study was undertaken to determine the pattern of MYCN protein (pMYCN) phosphorylation in human neuroblastoma cells. We report that pMYCN is phosphorylated in vitro by purified casein kinase II (CK-II). Two-dimensional phosphopeptide maps showed that most of the phosphopeptides of pMYCN phosphorylated in vitro by CK-II correspond to those phosphorylated in vivo. Fine mapping of the phosphorylation sites was performed using two synthetic MYCN peptides corresponding to pMYCN CK-II consensus sequences. Both peptides were found to be phosphorylated by CK-II and competitively inhibited CK-II phosphorylation of pMYCN in vitro. Thus, we have localized two major CK-II phosphorylation sites in pMYCN, one to the highly acidic central region and the second to serine 367 proximal to the C-terminus. Our data demonstrate that pMYCN is a physiological substrate for CK-II and, since CK-II activity is stimulated in response to mitogens, CK-II phosphorylation of pMYCN may, therefore, represent one signal transduction pathway used by neuroblastoma cells. PMID: 1923500 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 276: Cell Growth Differ. 1991 Oct;2(10):511-8. Decrease of proliferation rate and induction of differentiation by a MYCN antisense DNA oligomer in a human neuroblastoma cell line. Negroni A, Scarpa S, Romeo A, Ferrari S, Modesti A, Raschella G. Division of Physics and Biochemical Sciences, Ente per le nuove technologie per l'energia e l'ambiente (ENEA), Rome, Italy. The effects of an antisense oligodeoxynucleotide to codons 2-7 of the oncogene MYCN on the human neuroblastoma cell line LAN-5 were studied. Treated cells showed a decreased MYCN protein expression and synthesis by immunoperoxidase staining and immunoprecipitation. At the same time, the replication rate was inhibited, and the phenotype was modified toward a more differentiated type. Our data suggest the involvement of oncogene MYCN in both proliferative and differentiative processes. PMID: 1751406 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 277: Biochem Biophys Res Commun. 1991 Sep 30;179(3):1449-54. Enhanced MYCN oncogene expression in human neuroblastoma cells is associated with altered FGF receptor expression and cellular growth response to basic FGF. Schweigerer L, Ledoux D, Fleischmann G, Barritault D. Sektion Onkologie/Immunologie, Universitats-Kinderklinik, Ruprecht-Karls-Universitat, Heidelberg, FRG. We have examined the effect of human basic fibroblast growth factor (bFGF) on the proliferation of human neuroblastoma cells with normal and enhanced MYCN oncogene expression. bFGF stimulated the proliferation of the neuroblastoma cells with enhanced, but not normal, MYCN expression. Both cell species express FGFR-1, but not FGFR-2, receptors and both harbor FGF receptor species of Mr 145.000, but they differ in their pattern of lower and higher-molecular weight FGF receptor species. Our results demonstrate that enhanced MYCN expression confers to neuroblastoma cells the ability to respond to bFGF, possibly by inducing functional FGF receptors. This mechanism may contribute to the advanced malignant phenotype of human neuroblastomas with enhanced MYCN expression. PMID: 1656953 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 278: J Natl Cancer Inst. 1991 Aug 7;83(15):1085-8. Nonrandom distribution of N-myc oncogene genotypes in neuroblastoma. Waber PG, Bowcock AM, Arencibia-Mireles O, Nisen PD. Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas 75235-9063. The distributions of Pvu II and Sph I alleles of the N-myc oncogene (also known as MYCN) were studied in a series of normal individuals and pediatric patients with solid tumors. In the case of Pvu II, where the polymorphic site is located 3' of the gene, the frequencies of the allele were 0.27 (11-kilobase fragment) and 0.73 (8-kilobase fragment) in 43 unrelated normal Caucasians. The frequencies of the allele were similar in 40 non-N-myc-amplified neuroblastomas, 47 Wilms' tumors, and 31 other pediatric tumors. In these cases, the genotypes were in Hardy-Weinberg equilibrium. In 18 N-myc-amplified neuroblastomas, however, the observed genotype frequencies deviated from Hardy-Weinberg equilibrium (P less than .005). Similar observations were made with an Sph I restriction fragment length polymorphism where the polymorphic site is located in intron 2. The differences between amplified and nonamplified neuroblastomas suggest a possible involvement of sequences at or near N-myc in the progression of tumors where the N-myc gene is amplified. PMID: 1678788 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 279: Klin Padiatr. 1991 Jul-Aug;203(4):319-22. [Increased expression of the MYCN oncogene in human neuroblastoma cells and possible, new therapeutic approaches] [Article in German] Schweigerer L, Fotsis T. Sektion Onkologie/Immunologie, Universitats-Kinderklinik, Ruprecht-Karls-Universitat Heidelberg. Human neuroblastomas of advanced stages often display amplification with a consecutive enhanced expression of the MYCN oncogene. Enhanced MYCN expression is thought to contribute in a causative manner to the progression of neuroblastomas, but the mechanisms by which this may occur have remained unclear. By transfecting human neuroblastoma cells that display a normal MYCN expression with the human MYCN oncogene, we have generated a cell line with enhanced MYCN expression and thereby were able to compare the biological and biochemical properties of the transfected and non-transfected cells. We have demonstrated autocrine growth factors in the MYCN-transfected, but not the non-transfected, neuroblastoma cells. Identification of the primary structures of these factors may help to develop specific antagonists in order to improve the therapy of advanced neuroblastomas. Currently, this could be done by application of genistein or tumor necrosis factor. As we could demonstrate for the first time, the dietary constituent genistein is able to inhibit the proliferation of neuroblastoma cells with enhanced and normal MYCN expression, but also that of cells derived from other solid pediatric tumors. In contrast, tumor necrosis factor is able to inhibit selectively the proliferation of neuroblastoma cells with enhanced MYCN expression. We suggest that tumor necrosis factor might improve the therapy of advanced human neuroblastomas. PMID: 1942938 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 280: Int J Cancer. 1991 Jun 19;48(4):502-10. Expression of leucocyte adhesion molecules on 66 clinical neuroblastoma specimens. Favrot MC, Combaret V, Goillot E, Tabone E, Bouffet E, Dolbeau D, Bouvier R, Coze C, Michon J, Philip T. Centre Leon Berard, Lyon, France. LFA-3, ICAM-1, HLA.ABC and HLA.DR expression was analyzed on 66 neuroblastoma specimens. HLA.ABC was expressed on 26 specimens, HLA.DR on 2, LFA-3 on 20 and ICAM-1 on 10. HLA.ABC and LFA-3 were positive on ganglioneuroblastoma or ganglioneuroma, but they were negative on neuroblastoma, independently of the clinical staging; HLA.ABC and LFA-3 were induced in vivo by chemotherapy in parallel with tumoral cell differentiation, in both the primary and the metastases. The expression of ICAM-1 was restricted to 5 of the 10 low-grade stage-1 or stage-2 specimens, 1 stage-3 specimen, and the primary tumors of 2 patients with stage-4 disease, analyzed hence at diagnosis and after chemotherapy (4 specimens); metastatic cells obtained in 1 of these patients were negative. HLA.ABC and LFA-3 expressed on both mycN-negative and -positive specimens, whereas ICAM-1 was restricted to MYCN-negative specimens. LFA-3 diffusely stained partially differentiated neuroblasts, Schwann cells and ganglion cells. The expression of HLA.ABC on differentiated neuroblasts varied from one sample to another and within the same tumor; Schwann cells were strongly positive, but ganglion cells were negative. In positive samples, ICAM-1 was expressed on differentiated neuroblasts and Schwann cells, but negative on ganglion cells; however, most of the differentiated tumors were ICAM-1-negative, suggesting ICAM-1 induction by unknown local signal. The 4 markers were negative on undifferentiated neuroblasts. The distribution of these 4 markers on clinical specimens was in agreement with their reactivity on fetal tissues, as well as with results obtained on neuroblastoma cell lines before and after in vitro treatment with IFN-gamma. PMID: 1710608 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 281: Oncogene. 1991 Jun;6(6):969-77. Localization of regulatory elements controlling human MYCN expression. Hiller S, Breit S, Wang ZQ, Wagner EF, Schwab M. Institut fur Experimentelle Pathologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany. Expression of the cellular oncogene MYCN is restricted to few cell lineages and is highest during development both in mouse and in humans. In pursuit of elucidating the mechanisms underlying MYCN regulation we introduced a human MYCN clone (pNb-9) with approximately 2.5 kbp 5'- and 6 kbp 3'-flanking genomic sequences into different murine and human cell lines as well as into mice. In all cases we found a correlation between the expression of the exogenous and the endogenous MYCN. Among cell lines, only those expressing the endogenous gene also expressed the transfected gene. In the transgenic mice transcripts of the transgene were present in proportion to the transcripts of the endogenous MYCN gene with the highest level in the brain. Therefore, the genetic information necessary for regulated expression of MYCN appears to be contained in pNb-9. To localize the DNA-regions responsible for regulated expression, we generated MYCN/CAT hybrid genes with different portions of the putative MYCN promoter region linked to the reporter gene. Transient transfections into various murine and human cell lines identified three DNA regions apparently involved in the regulation of expression. One region about 200 bp upstream of the transcriptional start site is responsible for a basal level of MYCN expression. A second region located about 800 bp further upstream appears to be involved in cell type-specific gene activation. The third regulatory element is located at the 3' end of the first exon and/or in the first intron and may mediate tissue-specific down regulation of gene expression. PMID: 2067849 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 282: Br J Cancer. 1991 Jun;63(6):851-8. Oncogenes in human testicular cancer: DNA and RNA studies. Peltomaki P, Alfthan O, de la Chapelle A. Department of Medical Genetics, University of Helsinki, Finland. Oncogene dosage and expression were studied in 16 testicular neoplasms, 14 of germ cell and two of non-germ cell origin. In comparison with normal DNA, tumour DNA of a total of eight patients (seven with germ cell neoplasm and one with testicular lymphoma) showed increased dosages of KRAS2, PDGFA, EGFR, MET and PDGFB. The most frequent (occurring in six tumours) and prominent (up to 3-4-fold) increases were detected in the dosages of KRAS2 (on chromosome 12p) and PDGFA (chromosome 7p), relative to a reference locus from chromosome 2. Importantly, there was a similar increase in 12p dosage in general in these tumours, suggesting the presence of the characteristic isochromosome 12p marker. On the contrary, possible 7p polysomy (assessed by molecular methods) did not explain the PDGFA (or EGFR) changes in all cases. NRAS, MYCN, CSFIR, MYB, MYC, ABL, HRASI, TP53, and ERBB2 did not reveal any consistent alterations in tumour DNA. In RNA dot blot assays the expression of KRAS2, PDGFA, EGFR, or MYC was generally not increased in the tumour samples when compared to that in normal testicular tissue of the same patients although there was interindividual variation in mRNA levels. It thus appears that while oncogene dosage changes occur in a proportion of testis cancers, they are often part of changes in large chromosomal regions or whole arms and are seldom accompanied by altered expression. PMID: 1829952 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 283: Prog Clin Biol Res. 1991;366:71-6. Yeast artificial chromosome (YAC) vector cloning of the MYCN amplified domain in neuroblastomas. Schneider SS, Zehnbauer BA, Vogelstein B, Brodeur GM. Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110. About 25% of human neuroblastomas have amplification of the MYCN proto-oncogene, and this feature is associated with advanced stages of disease and rapid tumor progression. Estimates of the size of the amplicon range from 300 to 3,000 kb, with MYCN at or near the center. It has been determined previously that there is considerable conservation of the amplified sequences among different neuroblastomas, and very few rearrangements have been found. We decided to clone the entire amplified domain in YACs to assess the size, structure and organization of the amplified sequences in neuroblastomas and to identify novel or junctional sequences at the ends of an amplicon in double minutes (dmins) or in the germline. A YAC library was constructed from the SMS-KAN cell line, which contains dmins and has about 150 copies of MYCN. About 5,000 clones from this library were screened with MYCN as well as other probes from the amplified domain. To date, 16 YACs have been identified, with a median size of 240 kb (range 50-460 kb). These YAC clones can be arranged in a contiguous, linear map of greater than or equal to 1 megabase. Our data suggest that most neuroblastomas with MYCN amplification also amplify a region that is a megabase or more in size, making this the largest amplified domain cloned to date. The very large size suggests that there are other genes near MYCN whose expression is important in mediating the aggressive phenotype associated with MYCN amplification, or, alternatively, that the nearest origin of replication may be a considerable distance from MYCN. PMID: 2068181 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 284: Prog Clin Biol Res. 1991;366:151-6. Patterns of regulation of nuclear proto-oncogenes MYCN and MYB in retinoic acid treated neuroblastoma cells. Thiele CJ. Molecular Genetics Section, National Cancer Institute, Bethesda, MD 20892. PMID: 2068135 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 285: Prog Clin Biol Res. 1991;366:11-9. Inverse expression of MYCN and mdr-1 in human neuroblastoma. Nakagawara A, Kadomatsu K, Sato S, Kohno K, Takano H, Kuwano M. Department of Pediatric Surgery, Faculty of Medicine, Kyushu University, Fukuoka, Japan. PMID: 2068131 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 286: Adv Enzyme Regul. 1991;31:329-38. Enhanced expression of the cellular oncogene MYCN and progression of human neuroblastoma. Schwab M. Institute for Experimental Pathology, German Cancer Research Center, Heidelberg. A central issue in cancer research is how tumors evolve and acquire a more aggressive phenotype. It is a widely discussed hypothesis that tumor cell populations progress by evolutionary change as a result of the generation of a variant cell through genomic instability followed by selection of particular variant clones having a growth advantage within the particular tissue environment. Genetic instability appears to be characteristic of neoplastic cells, but no consistent increase in instability seems to accompany progression of the malignant phenotype of the tumor. It is reasonable to assume that quantitative or qualitative changes of cellular oncogenes contribute to the emergence of more malignant phenotypes. Although any one of the molecular changes of cellular oncogenes identified over the past years is a good candidate as an element in progression, amplification appears particularly frequently as a correlate to advanced tumor stage. The fact that amplification does not show up in all progressing tumors of a particular type, for instance in only 50% of advanced-stage neuroblastomas, is often construed as speaking against a role in progression. One should be aware, however, that it is the enhanced expression of a gene consequent to amplification and not amplification per se that affects the cellular phenotype. There are alternative molecular pathways by which expression of a particular gene may become deregulated. During the past decade much information has accrued about genetic alterations in tumor cells. The activation of the oncogenic potential of cellular genes can take different routes among which mutational alteration, translocation and amplification predominate. In particular, amplification has found its way to practical use due to its association with more aggressively growing types of human cancer. MYCN amplification in neuroblastoma is a paradigm for the prognostic significance of oncogene alteration, and at the same time has represented the clinical debut of oncogene research. The full significance of oncogene amplification as a predictor for poor prognosis became clear with the more recent identification of amplified ERBB2 in aggressively growing breast cancers. The state of the art is that amplified cellular oncogenes define cancer patients who have a poor prognosis and require a specific therapeutic regimen. Publication Types: Review Review, Tutorial PMID: 1877394 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 287: Bone Marrow Transplant. 1991;7 Suppl 3:133-5. MYCN amplification does not affect survival of neuroblastoma patients treated with autologous bone marrow transplantation. Sansone R, Di Martino D, Lanino E, Dini G, Massimo L, Tonini GP. Popul. Genetics Sect., Natl. Cancer Inst., Genova, Italy. From an extended series neuroblastoma cases evaluated for MYCN amplification (MNA) at the "G. Gaslini" Hospital 15 (4 with and 11 without NMA) underwent myeloablative therapy and bone marrow transplantation (MAT-ABMT). Such cases ranged in age at diagnosis from 13 months to 7 years and were followed up at least 8 months after MAT-ABMT. MNA was present in 2/10 cases dead for disease, in 0/1 cases alive with disease, and in 2/4 cases presently in complete clinical remission. This preliminary evidence would discourage to consider MNA as a marker capable of predicting the final outcome of patients with metastatic Nb. PMID: 1855077 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 288: Prog Clin Biol Res. 1991;366:37-43. Degradation of MYCN oncoprotein by the ubiquitin system. Ciechanover A, DiGiuseppe JA, Schwartz AL, Brodeur GM. Faculty of Medicine, Department of Biochemistry Technion-Israel Institute of Technology, Haifa. Nuclear oncoproteins are among the most rapidly degraded intracellular proteins. Previous work has implicated the ubiquitin-mediated proteolytic system in the turnover of short-lived intracellular proteins as a class. In the present study, we have evaluated the potential role of the ubiquitin system in the degradation of the specific nuclear oncoproteins encoded by the MYCN gene. The oncoproteins were synthesized in vitro by transcription of an MYCN cDNA, and translation of the resulting single species of mRNA in the presence of 35S-methionine. Degradation of labeled proteins was monitored in a cell-free system derived from rabbit reticulocytes. ATP stimulated the degradation of the MYCN protein more than 10 fold. ATP-dependent degradation was completely inhibited by neutralizing antibody directed against E1, the first enzyme in the ubiquitin-mediated proteolytic cascade. Moreover, ATP-dependent degradation in E1-depleted lysate could be restored by the addition of affinity-purified E1. These data suggest that the ubiquitin system mediates the ATP-dependent degradation of MYCN oncoproteins in vitro, and that these proteins possess signals that target them for rapid turnover by this proteolytic pathway. Although MYCN in neuroblastomas is activated primarily by amplification, it may be activated by other mechanisms in aggressive tumors with a single copy of MYCN. Mutations in protein coding sequence that alter the signals for recognition by protein degradation systems represent a potential mechanism by which oncogene activation might occur. PMID: 1648744 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 289: J Natl Cancer Inst. 1990 Dec 5;82(23):1815-21. Ability of circular extrachromosomal DNA molecules to carry amplified MYCN proto-oncogenes in human neuroblastomas in vivo. VanDevanter DR, Piaskowski VD, Casper JT, Douglass EC, Von Hoff DD. Tumor Institute, Swedish Hospital Medical Center, Seattle, Wash. Amplification of the proto-oncogene MYCN (also known as N-myc) in neuroblastomas has been shown to correlate with both disease stage and prognosis, yet little is known about the DNA structures that carry amplified MYCN genes in neuroblastomas in vivo. We have used DNA irradiation and pulsed-field gel electrophoresis to analyze MYCN amplification structures in eight neuroblastomas from separate patients (four primary tumors and four metastatic lesions exhibiting MYCN amplification). Six of the eight neuroblastomas (three primary tumors and three metastatic lesions) exhibited MYCN DNA irradiation profiles consistent with the presence of circular extrachromosomal DNA amplification structures. Five neuroblastomas possessed amplification structures within the size range of double minute chromosomes, and one contained smaller DNA circles. Two neuroblastomas exhibited MYCN DNA irradiation patterns consistent with larger (presumably chromosomal) amplification structures. Multiple sizes of DNA circles were observed in the neuroblastomas of four different patients, implying in vivo multimerization of amplification structures. The presence of circular MYCN amplification structures in six of eight neuroblastomas examined suggests that circular DNA molecules are important structures in in vivo gene amplification. PMID: 2250296 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 290: Brain Pathol. 1990 Sep;1(1):47-54. Neuroblastoma--clinical applications of molecular parameters. Brodeur GM. Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110. At least two genetic events have been identified which are characteristic of certain neuroblastomas. These are loss of a critical region on the short arm of chromosome 1, and amplification of the MYCN proto-oncogene. Our studies suggest that the two genetic events may be related, and that loss of heterozygosity (LOH) for chromosome 1p may precede the development of amplification. RAS gene mutations appear to be rare, and no other oncogene has been shown to be consistently activated by amplification or other mechanism. LOH for chromosome 14q has been identified recently, but its frequency and significance is not clear. Based on flow cytometric analysis of DNA content, tumour cytogenetics and molecular studies, three distinct genetic subsets of neuroblastomas are emerging. The first is characterized by a hyperdiploid or near-triploid modal karyotype, with few if any cytogenetic rearrangements. These patients are generally less than one year of age with localized disease and a good prognosis. The second group is characterized by a near-diploid or near-tetraploid karyotype, with no consistent rearrangement identified to date. They are generally older patients with more advanced stages of disease that progress slowly and are ultimately fatal. The third group is characterized by a near-diploid or tetraploid karyotype, with deletions or LOH for chromosome 1p, amplification of MYCN, or both. These patients are generally older with advanced stages of disease which is rapidly progressive. Thus, genetic analysis of neuroblastoma cells by karyotype, flow cytometry and determination of MYCN copy number provides information that has prognostic significance and can more appropriately direct the choice of treatment. A better understanding of these genetic abnormalities and the biochemical pathways that they effect may provide insights into malignant transformation and progression, as well as provide targets for future therapeutic approaches. Publication Types: Multicenter Study Review Review, Tutorial PMID: 1669693 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 291: Brain Pathol. 1990 Sep;1(1):41-6. Amplification of the MYCN oncogene and deletion of putative tumour suppressor gene in human neuroblastomas. Schwab M. Institute for Experimental Pathology, German Cancer Research Center, Heidelberg. Human neuroblastoma cells often carry non-random chromosomal abnormalities signalling genetic alterations. Quite frequent are 'double minutes' (DMs) and homogeneously staining regions (HSRs), both cytogenetic manifestations of amplified DNA, and chromosome 1p-deletions indicating loss of genetic information. With the identification of amplified MYCN and the demonstration of a consensus deletion spanning the chromosome 1p36.1-2 region it appears now likely that both amplification of a cellular oncogene and loss of a tumour-suppressor gene play an important role in neuroblastoma. Amplification of MYCN is an indicator for poor prognosis, even when classical morphological criteria would suggest a better outcome. Consequently, patients with amplification are subjected to more intensive therapeutic regimens. Amplification of MYCN is a paradigm for the clinical use of an oncogene alteration. Publication Types: Review Review, Tutorial PMID: 1669692 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 292: Biochem Biophys Res Commun. 1990 Aug 16;170(3):1301-7. Enhanced MYCN oncogene expression in human neuroblastoma cells results in increased susceptibility to growth inhibition by TNF alpha. Schweigerer L, Scheurich P, Fotsis T. Sektion Onkologie/Immunologie, Universitat-Kinderklinik Ruprecht-Karls-Universitat, Heidelberg, FRG. Human neuroblastoma cells with normal expression of the endogenous MYCN oncogene were transfected with a vector containing an exogenous MYCN gene. The transfected cells expressed the exogenous MYCN at high levels and had acquired a phenotype resembling that of cells from advanced human neuroblastomas. Proliferation of the MYCN-transfected, but not of the untransfected, neuroblastoma cells was inhibited by low concentrations of recombinant human tumor necrosis factor alpha (TNF alpha). Our results suggest that TNF alpha could be useful for the treatment of advanced human neuroblastomas, in which high MYCN expression seems to be a causative factor. PMID: 2202299 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 293: Klin Padiatr. 1990 Jul-Aug;202(4):197-201. [Amplification of N-myc in neuroblastoma: paradigm for clinical use of an oncogene alteration] [Article in German] Schwab M. Institut fur Experimentelle Pathologie, Deutsches Krebsforschungszentrum. Increase of the dosage of cellular oncogens by DNA amplifications is a frequent genetic alteration of cancer cells. The presence of amplified cellular oncogenes is usually signalled by conspicuous chromosomal abnormalities, "double minutes" (DMs) or "homogeneously staining regions (HSRs). Some human cancers carry a specific amplified oncogene at high incidence. Particularly in neuroblastomas and in breast cancers the amplification of cellular oncogenes has been found associated with aggressively growing cancers and is an indicator for poor prognosis. Neuroblastoma, a malignant tumor of the sympathetic nervous system of children, frequently carries amplification of the oncogene MYCN. The amplification of MYCN is of predictive value for identifying high risk neuroblastoma patients that require specific therapeutic regimen and is generally viewed as the first oncogene alteration that turned out to be of practical clinical significance. Publication Types: Review Review, Tutorial PMID: 2203936 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------