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At least 19 recordsLinked to original sources
PubMed2006

The MYCN enigma: significance of MYCN expression in neuroblastoma.

MYCN amplification strongly predicts adverse outcome of neuroblastoma. However, the significance of MYCN expression in the clinical and biological behavior of neuroblastoma has been unclear. To address this question, we first examined the expression of MYCN in combination with TrkA (a favorable prognostic indicator of neuroblastoma) in 91 primary neuroblastoma by quantitative reverse transcription-PCR and investigated the relationship among patient survival, MYCN, and TrkA expressions. Three subsets of neuroblastoma were defined based on MYCN and TrkA expression. Neuroblastoma expressing the highest level of MYCN but little TrkA were MYCN-amplified cases, which had a 5-year survival of 9.3%. Interestingly, MYCN and TrkA expression showed a linear correlation (r = 0.5664, P < 0.00005) in neuroblastoma lacking MYCN amplification, and the 5-year survival of neuroblastoma patients with low MYCN and low TrkA expressions was 63.7%, whereas those with high expression of both had a 5-year survival of 88.1% (P < 0.00005). This nonlinear distribution of disease outcome relative to MYCN expression in neuroblastoma explains why MYCN expression is not predictive of neuroblastoma disease outcome by dichotomous division of the neuroblastoma cohort. However, high-level MYCN expression is associated with favorable outcome in neuroblastoma lacking MYCN amplification. Furthermore, forced expression of MYCN significantly suppresses growth of neuroblastoma cells lacking MYCN amplification by inducing apoptosis and enhancing favorable neuroblastoma gene expression. Collectively, these data suggest that high-level MYCN expression in neuroblastoma lacking MYCN amplification results in a benign phenotype. Thus, the high MYCN expression confers the opposite biological consequence in neuroblastoma, depending on whether or not MYCN is amplified.

Age Factors↗
PubMed2000

MYCN expression is not prognostic of adverse outcome in advanced-stage neuroblastoma with nonamplified MYCN.

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.

Blotting, Western↗
PubMed2004

Mechanisms of embryonal tumor initiation: distinct roles for MycN expression and MYCN amplification.

The mechanisms causing persistence of embryonal cells that later give rise to tumors is unknown. One tumorigenic factor in the embryonal childhood tumor neuroblastoma is the MYCN protooncogene. Here we show that normal mice developed neuroblast hyperplasia in paravertebral ganglia at birth that completely regressed by 2 weeks of age. In contrast, ganglia from MYCN transgenic (TH-MYCN) mice demonstrated a marked increase in neuroblast hyperplasia and MycN expression during week 1. Regression of neuroblast hyperplasia was then delayed and incomplete before neuroblastoma tumor formation at 6 and 13 weeks in homo- and hemizygote mice, respectively. Paravertebral neuronal cells cultured from perinatal TH-MYCN mice exhibited 3- to 10-fold resistance to nerve growth factor (NGF) withdrawal, compared with normal mice. Both low- and high-affinity NGF receptors were expressed in perinatal neuroblast hyperplasia but not in neuroblastoma tumor tissue. MYCN transgene amplification was present at low levels in perinatal neuroblast hyperplasia from both homo- and hemizygote TH-MYCN mice. However, only in hemizygous mice did tumor formation correlate with a stepwise increase in the frequency of MYCN amplification. These data suggest that inappropriate perinatal MycN expression in paravertebral ganglia cells from TH-MYCN mice initiated tumorigenesis by altering the physiologic process of neural crest cell deletion. Persisting embryonal neural crest cells underwent further changes, such as MYCN amplification and repression of NGF receptor expression, during tumor progression. Our studies provide a model for studying perinatal factors influencing embryonal tumor initiation.

Animals↗
PubMed2001

Rearrangement in the coding region of the MYCN gene in a subset of amplicons in a case of neuroblastoma with MYCN amplification.

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.

Abdominal Neoplasms↗
PubMed2000

Lysis of MYCN-amplified neuroblastoma cells by MYCN peptide-specific cytotoxic T lymphocytes.

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.

Adult↗
PubMed1996

Amplification of a DEAD box gene (DDX1) with the MYCN gene in neuroblastomas as a result of cosegregation of sequences flanking the MYCN locus.

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.

Amino Acid Sequence↗
PubMed1999

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.

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.

Carrier Proteins↗
PubMedDate not supplied

In vitro effects of MYCN sense and antisense expression in MYCN-amplified human neuroblastoma cells.

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.

Antineoplastic Agents↗
PubMed2006

The role of MYCN in the failure of MYCN amplified neuroblastoma cell lines to G1 arrest after DNA damage.

We previously reported that 3 p53 wild type (wt) MYCN amplified (MNA) neuroblastoma cell lines failed to G1 arrest after DNA damage despite induction of p53, p21(WAF1) and MDM2. We hypothesised that this was due to high MYCN expression. p53 responses to DNA damage were examined in an additional 13 p53 wt neuroblastoma cell lines. MNA was significantly associated with a failure to G1 arrest after DNA damage (p < 0.001) and higher levels of apoptosis after irradiation (p < 0.05). p21(WAF1) and hypophosphorylated (hypo) RB accumulation post irradiation were significantly lower in cell lines that failed to G1 arrest (p < 0.05). Conditional MYCN expression in non-MNA SHEP Tet21N cells did not affect the G1 arrest after irradiation. MYCN knockdown using siRNA in 3 p53 wt MNA cell lines did not restore a G1 arrest after irradiation, but increased the baseline G1 population, p21(WAF1) and hypo RB expression. MYCN siRNA also caused a G1 arrest in a p53 mutant MNA cell line. This study is the first to determine that MNA correlates with a failure to G1 arrest and attenuated p21(WAF1) induction; however MYCN expression alone is not causally responsible.

Apoptosis↗
PubMed1993

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.

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.

Alleles↗
PubMed2003

MYCN gain and MYCN amplification in a stage 4S neuroblastoma.

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.

Adrenal Gland Neoplasms↗
PubMed2006

The coamplification pattern of the MYCN amplicon is an invariable attribute of most MYCN-amplified human neuroblastomas.

PURPOSE: Fifteen percent to 20% of human neuroblastomas show amplification of the MYCN oncogene physiologically located at chromosome 2p24-25, indicating an aggressive subtype of human neuroblastoma with a poor clinical outcome. Recent findings revealed that the structure of the amplicon differs interindividually and that coamplification of genes in telomeric proximity to MYCN might play a relevant role in neuroblastoma development and response to treatment, respectively. We now asked if the amplicon structure is an invariable attribute of an individual tumor or if the coamplification pattern could change during progress or in case of recurrent disease. EXPERIMENTAL DESIGN: We used a previously described multiplex PCR approach to analyze the coamplification status of MYCN-amplified human neuroblastomas (n = 33) in tumor tissue at the time of initial diagnosis and in consecutive tissue specimens at later time points after initial treatment or from relapsing disease. The MYCN copy number per haploid genome (Mcn/hg) in these specimens was determined in a separate duplex PCR. RESULTS: In 32 of the 33 investigated tumors, the amplicon structure showed no changes after initial chemotherapy and in recurrent disease. Mcn/hg showed a decrease after initial treatment (n = 23), whereas we found a significant increase in recurrent disease (n = 10). CONCLUSION: Our data indicate that the initial determined structure of the 2p24-25 amplicon is a consistent attribute in the great majority of the individual MYCN-amplified neuroblastomas and shows no plasticity during or after chemotherapy. Observed changes in the Mcn/hg over the course of disease are in line with preexisting cell culture findings.

Adolescent↗
PubMed2005

Key role for p27Kip1, retinoblastoma protein Rb, and MYCN in polyamine inhibitor-induced G1 cell cycle arrest in MYCN-amplified human neuroblastoma cells.

Alpha-difluoromethylornithine (DFMO) inhibits the proto-oncogene ornithine decarboxylase (ODC) and is known to induce cell cycle arrest. However, the effect of DFMO on human neuroblastoma (NB) cells and the exact mechanism of DFMO-induced cell death are largely unknown. Treatment with DFMO in combination with SAM486A, an S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor, has been shown to enhance polyamine pool depletion. Therefore, we analysed the mechanism of action of DFMO and/or SAM486A in two established MYCN-amplified human NB cell lines. DFMO and SAM486A caused rapid cell growth inhibition, polyamine depletion, and G1 cell cycle arrest without apoptosis in cell lines LAN-1 and NMB-7. These effects were enhanced with combined inhibitors and largely prevented by cotreatment with exogenous polyamines. The G1 cell cycle arrest was concomitant with an increase in cyclin-dependent kinase inhibitor p27Kip1. In a similar fashion, DFMO and DFMO/SAM486A inhibited the phosphorylation of the G1/S transition-regulating retinoblastoma protein Rb at residues Ser795 and Ser807/811. Moreover, we observed a dramatic decrease in MYCN protein levels. Overexpression of MYCN induces an aggressive NB phenotype with malignant behavior. We show for the first time that DFMO and SAM486A induce G1 cell cycle arrest in NB cells through p27Kip1 and Rb hypophosphorylation.

Adenosylmethionine Decarboxylase↗
PubMed2002

Intensified chemotherapy increases the survival rates in patients with stage 4 neuroblastoma with MYCN amplification.

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.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2005

Enlarged and prominent nucleoli may be indicative of MYCN amplification: a study of neuroblastoma (Schwannian stroma-poor), undifferentiated/poorly differentiated subtype with high mitosis-karyorrhexis index.

BACKGROUND: According to the International Neuroblastoma Pathology Classification, neuroblastomas exhibiting MYCN amplification (A-MYCN) have unique histologic features-namely, undifferentiated/poorly differentiated subtype with a high mitosis-karyorrhexis index (U/PD-H). Nonetheless, certain tumors possessing these histologic characteristics contain a nonamplified MYCN gene (NA-MYCN). METHODS: The clinical characteristics of patients from the Children's Cancer Group (CCG) 3881 and 3891 studies who had neuroblastoma, U/PD-H, exhibiting A-MYCN (n=68) or NA-MYCN (n=33) were investigated. The histologic and cytologic features of tumors (A-MYCN, n=62; NA-MYCN, n=28) filed at the Pathology Reference Laboratory, Department of Pathology and Laboratory Medicine, Childrens Hospital Los Angeles, were reviewed, and nucleolar areas in undifferentiated neuroblastic cells were evaluated using image analysis methods. RESULTS: All 68 patients whose tumors exhibited A-MYCN had disease that was in an advanced clinical stage (Stage III or IV); 89.7% of these patients were diagnosed between ages 0.5 and 3.5 years, and 67 of the 68 had been treated with the high-risk protocol in the CCG-3891 study. Children whose tumors exhibited NA-MYCN were evenly distributed across all age groups; 30 of these 33 children had advanced-stage disease, and 26 had been treated with a high-risk protocol. The prognosis associated with A-MYCN (event free survival [EFS], 15.7%; overall survival [OS], 22.2%) was significantly poorer than the prognosis associated with NA-MYCN (EFS, 56.1%; OS, 69.3%). The lone histologic/cytologic difference between tumors exhibiting A-MYCN and tumors exhibiting NA-MYCN involved nucleolar appearance. Neuroblastic cells in tumors exhibiting A-MYCN were characterized by the presence of 1 or more large, prominent nucleoli, and the mean nucleolar area was significantly greater in the 18 tumors exhibiting A-MYCN that were assessed (7.63 microm2) than in the 16 tumors exhibiting NA-MYCN that were assessed (5.53 microm2; P=0.004). CONCLUSIONS: Neuroblastomas, U/PD-H, were found to vary in terms of molecular background and clinical behavior. The results of the current study indicate that nucleolar enlargement in neuroblastic cells may be a sign of MYCN amplification.

Biomarkers, Tumor↗
PubMed2004

Clinical significance of a highly sensitive analysis for gene dosage and the expression level of MYCN in neuroblastoma.

BACKGROUND: The amplification of the MYCN gene is one of the most powerful adverse prognosis factors in neuroblastoma, but the clinical significance of an enhanced expression of MYCN remains controversial. To reassess the clinical implications of MYCN amplification and expression in neuroblastoma, the status of amplification and the expression level of the MYCN gene of primary neuroblastoma samples were analyzed using highly sensitive analyses. METHODS: Using a quantitative polymerase chain reaction (PCR) method (TaqMan), the gene dosages (MYCN/p53) of 66 primary neuroblastoma samples were determined. In all 66 samples, the status of MYCN amplification has been determined previously by the Southern blotting method. Of the 54 samples with a single copy of MYCN based on the Southern blotting method, 23 samples were analyzed for MYCN amplification using the fluorescence in situ hybridization (FISH) method. The expression levels (MYCN/GAPDH) of 56 samples were determined by a quantitative reverse transcriptase (RT)-PCR method. RESULTS: Of the 54 samples with a single copy of MYCN based on the Southern blotting method, 46 samples showed MYCN gene dosages of less than 2.0, whereas the remaining 8 samples with dosages of more than 2.0 were tumors from patients with advanced-stage disease. The results of FISH supported the fact that these 8 samples contained a small number of MYCN-amplified cells. The cases of MYCN gene dosages of more than 2.0 were significantly associated with all other unfavorable prognostic factors (an age of >1 year at diagnosis [P <.0001], nonmass screening [P =.0003], advanced stage [P <.0001], diploid or tetraploid [P <.0001], and a Shimada unfavorable histology [P <.0001]). MYCN gene dosages of more than 2.0 were significantly associated with a high expression of MYCN (P =.0459). However, the expression level of MYCN was not significantly associated with any other prognostic factors. CONCLUSIONS: Quantitative PCR may thus be a useful modality for performing a highly sensitive and accurate assessment of the amplification and expression levels of the MYCN gene. In particular, the combination of the quantitative PCR system and the FISH method is considered to be a highly effective method for evaluating the status of MYCN amplification. In this highly sensitive analysis, MYCN amplification (MYCN/p53 > or = 2.0) was reconfirmed to be a strongly unfavorable factor, whereas the expression level of MYCN does not appear to be an independently significant prognosis factor.

Child↗