Use of tumor-specific gene expression for the differential diagnosis of neuroblastoma from other pediatric small round-cell malignancies.

The differential diagnosis of neuroblastoma from other small round-cell tumors of childhood, although clinically of great importance, is sometimes difficult due to the almost indistinguishable appearance of such tumors by conventional microscopy. Because neuroblastomas are characterized by the synthesis of catecholamines, we investigated the possibility that expression of genes involved in this pathway could serve as a molecular marker for this disease. A reverse transcriptase polymerase chain reaction assay was used to analyze expression of tyrosine hydroxylase and dopa decarboxylase in 84 pediatric malignancies including 55 neuroblastomas, 6 Ewing's sarcomas/primitive neuroectodermal tumors, 7 lymphomas, 6 leukemias, 2 rhabdomyosarcomas, 6 osteosarcomas, and 2 phaeochromocytomas. Of the 55 neuroblastoma samples analyzed, 54 expressed clearly detectable levels of both genes. The one sample that did not express either of the genes was rediagnosed both clinically and by molecular genetic analysis as a Ewing's sarcoma. Of the 29 non-neuroblastoma tumor samples examined, the only tumor samples that expressed clearly detectable levels of both tyrosine hydroxylase and dopa decarboxylase were phaeochromocytomas. Like neuroblastomas, these tumors are characterized by high levels of catecholamines. These findings suggest that expression of genes involved in catecholamine biosynthesis may be useful for differentiating neuroblastoma from other small round-cell tumors of childhood.

Altered expression of the MYCN oncogene modulates MRP gene expression and response to cytotoxic drugs in neuroblastoma cells.

We have recently shown a close correlation between expression of the Multidrug Resistance-associated Protein (MRP) gene and the MYCN oncogene and provided evidence that high MRP expression is a powerful independent predictor of poor outcome in neuroblastoma (Norris et al., New Engl. J. Med., 334, 231-238, 1996). The effect of MYCN down-regulation on MRP expression and response to cytotoxic drugs was investigated in NBL-S neuroblastoma cells transfected with MYCN antisense RNA constructs. Concomitant with MYCN down-regulation, the level of MRP expression was decreased in the NBAS-4 and NBAS-5 antisense transfectants. These cells demonstrated significantly increased sensitivity to the high affinity MRP substrates vincristine, doxorubicin, sodium arsenate and potassium antimony tartrate, but not to the poor MRP substrates, taxol or cisplatin. Similarly, transfection of full-length MYCN cDNA into SH-EP neuroblastoma cells resulted in increased MRP expression and significantly increased resistance specifically to MRP substrates. The results provide evidence for the MYCN oncogene influencing cytotoxic drug response via regulation of MRP gene expression. Our data also provide a link between the malignant and chemoresistant phenotypes of this childhood malignancy.

Prognostic significance of MYCN oncogene expression in childhood neuroblastoma.

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.

Evidence that the MYCN oncogene regulates MRP gene expression in neuroblastoma.

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.

The prognostic value of MDR1 gene expression in primary untreated neuroblastoma.

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.

Expression of the gene for multidrug-resistance-associated protein and outcome in patients with neuroblastoma.

BACKGROUND: Overexpression of the gene for the multidrug-resistance-associated protein (MRP) has been linked with resistance to chemotherapeutic agents (multidrug resistance) in vitro. The expression of MRP by neuroblastoma cells correlates with N-myc oncogene amplification, a well-established prognostic indicator in patients with neuroblastoma. METHODS: To relate MRP gene expression to established prognostic markers and the clinical outcome of neuroblastoma, we analyzed MRP expression in specimens of primary tumors from 60 patients with neuroblastoma. RESULTS: Levels of MRP gene expression were significantly higher in tumors with N-myc amplification than in tumors without such amplification (P < 0.001). High levels of MRP expression were strongly associated with reductions in both survival and event-free survival (P < 0.001) in the overall study population and in subgroups of patients without N-myc amplification and patients with localized disease. For the overall study population, the five-year cumulative survival rates in the groups with high and low levels of MRP expression were 57 percent (95 percent confidence interval, 37 to 78 percent) and 94 percent (95 percent confidence interval, 86 to 100 percent), respectively. In contrast, expression of the MDR1 multi-drug-resistance gene was not predictive of survival or event-free survival. After adjustment by multivariate analysis for the effects of N-myc amplification and other prognostic indicators, high levels of MRP expression retained significant prognostic value for poor survival (relative hazard, 14.9; P = 0.01) and poor event-free survival (relative hazard, 9.7; P = 0.004), whereas N-myc amplification had no prognostic value. CONCLUSIONS: High levels of MRP gene expression in patients with neuroblastoma correlate strongly with poor outcome. The findings suggest that expression of this multidrug-resistance gene accounts for the association between N-myc amplification and reduced survival.

Expression of the multidrug resistance-associated protein (MRP) gene correlates with amplification and overexpression of the N-myc oncogene in childhood neuroblastoma.

The MRP gene (Cole et al., Science (Washington DC), 258: 1650-1654, 1992) encodes a membrane-bound glycoprotein the expression of which correlates with non-P-glycoprotein-mediated multidrug resistance in a variety of cultured human cell lines. Using an RNA-polymerase chain reaction assay, expression of this gene was examined in the highly chemoresistant pediatric malignancy, neuroblastoma. MRP expression was observed in 5 human neuroblastoma cell lines and in all 25 primary neuroblastoma tumors of stage I through IVS. Tumors with amplification of the N-myc oncogene were found to have significantly higher MRP expression that those with no amplification (P = 0.0016). Expression of the MRP gene in the tumor specimens was highly correlated with expression of the N-myc gene (P = 0.0009), while expression of the MDR1 gene, encoding P-glycoprotein, was not related to expression of either the N-myc or MRP genes. Decreased expression of the N-myc oncogene in neuroblastoma cell lines SH-SY5Y and BE(2)-C, following treatment with retinoic acid, was paralleled by down-regulation of MRP gene expression, contrasting with increased expression of the MDR1 gene. Expression of the MRP gene is thus common in both primary neuroblastoma tumors and cultured cell lines, and correlates with amplification and overexpression of the N-myc oncogene, which is central to the malignant phenotype of this disease.