ABSTRACT: Neuroblastoma is the most common extracranial solid tumor of childhood, accounting for 15% of cancer-related deaths. These tumors have a predilection for young children; 60% of cases occur before age 2 years and 97% before age 10. Neuroblastomas derive from embryonic neural crest cells of the peripheral sympathetic nervous system. The behavior of this malignancy is characterized by marked clinical heterogeneity, ranging from spontaneous maturation in some patients to inexorable rapid metastatic progression in others. This article will discuss some of the molecular and biological features of neuroblastoma that are associated with these differences in behavior, and how these features have been used to develop a risk-based approach to therapy. [ONCOLOGY 11(12):1857-1866, 1997]
Approximately 500 cases of neuroblastoma are diagnosed annually in the United States. Overall incidence from birth to age 15 is 8.7 cases per million per year. The disease has a striking predilection for young children; 60% of cases occur before age 2 years and 97% before age 10.
Thus far, no definite environmental risk factors have been determined, although single studies have suggested a possible increased risk from maternal exposure to alcohol, neurally active drugs, diuretics, and hair coloring and from paternal exposure to electromagnetic fields.
The clinical behavior of neuroblastoma varies markedly, ranging from spontaneous maturation in some patients to inexorable rapid metastatic progression in others. This article will explore some of the molecular and biological features of neuroblastoma that are associated with these differences in behavior and the use of these features to develop a risk-based approach to therapy.
Deletion of the distal short arm of chromosome 1 was the first described genetic mutation in neuroblastoma tumors and is the most consistently reported abnormality.[1,2] Cytogenetic analysis of near-diploid neuroblastoma tumors and cell lines shows deletion of the distal short arm of chromosome 1 in 70% of cases. This deletion is found more frequently in patients with advanced disease and a poor prognosis. However, due to the technical difficulties inherent in karyotyping solid tumors, fewer patients with low-stage disease have been examined, although those tested have lacked the abnormality.
To eliminate the need for karyotyping, Fong et al studied DNA by comparing constitutional (lymphocyte) DNA to tumor DNA. They identified loss of heterozygosity at one or more loci on chromosome 1 in 13 of 47 tumors and showed, again, that this abnormality correlates with advanced, poor-prognosis disease. Subsequent investigation revealed frequent deletions on chromosome 11 and chromosome 14, as well as rearrangements on chromosome 17.
More recent screening studies for abnormalities of chromosome number have used comparative genomic hybridization. This technique summarizes DNA copy number abnormalities by mapping them to their positions on normal metaphase chromosomes, based on simultaneous hybridization of tumor and normal DNA labeled in different colors to normal metaphase spreads. The ratio of tumor to normal fluorescence intensities along the target chromosomes indicates relative increases and decreases in DNA copy number throughout the entire tumor genome. In a preliminary study of 29 cases from the Children’s Cancer Group (CCG), Plantaz et al showed that chromosome 17 gains were the most frequent abnormality in neuroblastoma, seen ingreater than 70% of cases.
One of the other intriguing abnormalities found in neuroblastoma tumors, which has been shown to correlate very strongly with outcome, is MYCN amplification, which is found in about 30% of tumors. The MYCN proto-oncogene is derived from the c-myc viral oncogene and is located on the distal arm of chromosome 2. The finding of MYCN amplification correlates with the cytogenetic abnormalities of double minutes (small extrachromosmal amplifi- fications of MYCN DNA) and homogeneously staining regions (regions of MYCN-amplified DNA that can be integrated into any otherwise normal chromosome).
Amplification of MYCN has now been shown to correlate very closely with advanced-stage disease and, within each stage, to be highly predictive of outcome. MYCN may now readily be determined by traditional Southern blot DNA analysis, the technique of fluorescence in situ hybridization (FISH; Figure 1), immunostaining of tissues for protein expression, and the sensitive polymerase chain reaction (PCR), as well as by comparative genomic hybridization.[4-7]
Other genes commonly implicated in cancers, such as the ras family of p53, have not been commonly affected in neuroblastoma tumors.
Chromosomal ploidy is another marker of prognosis that has proved to be particularly useful in infants less than 18 months of age. Near-diploid or pseudodiploid tumors have near-normal nuclear DNA content but often have structural chromosomal aberrations, including MYCN amplification. In contrast, hyperdiploid or near-triploid tumors are most common in infants who have tumors that lack 1p deletion or MYCN amplification and have an excellent prognosis. A small subset of infants with diploid tumors have a much poorer prognosis than those with hyperdiploidy.
Cellular Markers of Differentiation
Several cellular markers of differentiation along neuronal pathways have been shown to have prognostic importance in neuroblastoma, suggesting that the malignant transformation of these cells may result, in part, from inadequate response to the usual inducers of neuronal differentiation. Investigations have demonstrated abnormalities in the nerve growth factor receptors in neuroblastoma cell lines. These studies have shown a significant correlation between the increased expression of high-affinity nerve growth factor receptor (gp140TRK-A) and tumors with single-copy MYCN and a favorable outcome, as well as a correlation between lack of expression of the nerve growth factor receptors and both MYCN amplification and poor survival.
Another determinant of malignant progression may be telomerase, an RNA-protein complex that prevents cell senescence by maintaining chromosomal telomeres. Telomerase is expressed in normal germ-line but not most somatic cells, and it appears to be necessary for immortalization of malignant cells.
Telomerase activity has recently been reported to correlate with both MYCN amplification and stage in neuroblastoma. We have now examined a large group of primary neuroblastomas for RNA levels of telomerase and have shown correlations with both stage and event-free survival, with poorer event-free survival in patients whose tumors show higher telomerase expression.
In addition to the many molecular genetic determinants of malignant progression, many more general laboratory, pathologic, and clinical markers have been shown to correlate with prognosis (Table 1). Shimada has developed a powerful classification system that combines age with histopathologic grading (based on the amount of neural stroma), mitotic-karyorrhectic index, and differentiation, as well as diffuse vs nodular appearance. This prognostic classification, which appears to be independent of MYCN amplification, has been verified in both retrospective and prospective CCG studies.[12,13]
A number of serum markers have also shown to be prognostic for outcome, probably because, in part, they are surrogate markers of tumor burden. These include serum levels of lactic dehydrogenase (LDH), ferritin, GD2 ganglioside, and chromogranin.
The most important clinical risk factors are disease stage and age at diagnosis, with age less than 1 year conferring the most favorable prognosis. Other clinical risk factors that may have some adverse importance include bone metastases, bone marrow metastases, and primary site in the abdomen (as opposed to the more favorable sites of the pelvis or thorax).[2,14-18]
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