Key words: neuroblastoma, pediatric tumors, prognosis, MYCN, NMYC, 1p, 17q, 11q, ploidy, chromosome, amplification, oncogene, tumor suppressor gene, genetic instability, apoptosis, silencing, methylation, caspase

Abstract

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, 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.

INTRODUCTION

The nervous system is the most common site for the development of solid tumors in young children. Neuroblastoma is a malignant tumor consisting of neural crest derived undifferentiated neuroectodermal cells. The yearly incidence of neuroblastoma is in the range of 1 case per 100 000 children under the age of 15 years, accounting for 8 to 10 percent of all childhood cancers. Neuroblastoma typically presents during infancy or toddler years. Some 90% of children with the disease are diagnosed within the first 5 years of life, typically characterizing an embryonic tumor.

Neuroblastoma is often described as enigmatic and unpredictable because it exhibits three distinct patterns of clinical behavior: life-threatening progression; maturation to ganglioneuroblastoma or ganglioneuroma; and spontaneous regression. Many neuroblastomas present at diagnosis with metastatic disease and are usually associated with poor survival despite intensive therapy. Approximately 40 percent of all patients with neuroblastoma belong to this high-risk group, and therapeutic improvements in the past decade have not substantially improved their outlook in concert with other pediatric malignancies[1, 2]. On the other hand, some tumors undergo complete spontaneous regression even without therapy. The incidence of spontaneous regression in neuroblastoma is between 10 and 100 fold greater than that for any other human cancer[3]. The most convincing demonstration of spontaneous regression is when primary neuroblastoma and metastatic disease disappear without any treatment. This phenomenon usually occurs as part of a clinically recognizable syndrome designated 4s: a small primary tumor localized in the adrenal gland is accompanied with metastasis in the liver and/or bone marrow and skin but not in the cortical bone or distant nodes. Although spontaneous regression mainly occurs in infants, it is well described in older patients[4, 5]. Maturation to benign ganglioneuroma is less frequent and usually observed after chemotherapy[2]. A systematic evaluation of ganglioneuroma frequency has been hampered by the lack of recording of these benign tumors in any of the worldwide tumor registries.
Neuroblastomas frequently produce increased levels of catecholamines whose metabolites (vanillylmandelic acid and homovanillic acid) are detectable in the urine. This has been the basis for extensive screening studies to identify patients with neuroblastoma earlier in the course of their disease. This assumes that patients with more advanced stages of disease and a higher risk of treatment failure might develop from more localized disease over time. Extensive experience in Japan, North America and Europe suggests that the incidence of neuroblastoma in screening populations has increased by approximately 2-fold over that seen in unscreened populations, whereas the incidence of neuroblastoma in patients with advanced stage disease over 1 year of age has not changed accordingly[6-8]. This implies that infants with low stage disease and good prognosis detected clinically are those who remain out of a much larger number in whom the neuroblatoma cells undergo apoptosis or maturation and are not detected clinically. It is questionable, however, whether advanced stage tumors do in fact develop from early stages, or whether they represent a subgroup. This extreme clinical heterogeneity has raised the question whether neuroblastoma may consist of at least two distinct clinical-biologic types [7], which may be distinguished at diagnosis by specific genetic features. Transition from one type to the other appears to occur rarely, if ever[9].
However, from the clinical perspective a precise prediction at diagnosis of tumor behavior would be desirable to avoid treatment failure. Currently, prognostic evaluation is based primarily on the degree of tumor spread at diagnosis and age of the patients, only more recently several biological markers have been incorporated[10]. Tumor histology describing the degree of ganglionic differentiation and the extent of Schwanninan stroma has been widely accepted to be of prognostic importance[11]. In addition, several genetic markers have been established which allow tumors to be classified into subsets with distinct biological features. The most widely accepted classification proposes three distinct but interrelated subsets based on genetic and biological features[9]. In fact, certain genetic alterations are strong predictors of response to therapy and outcome, and as such they are remarkably efficient at properly assigning patients to the appropriate intensity of therapy. This makes neuroblastoma a paradigm for the clinical importance of tumor genetic alterations. However, treatment failure occurs in all patient subgroups, which suggests that additional prognostic markers may be available to further refine treatment decision. In addition, research directed at sites of genetic alterations will provide insights into mechanisms of malignant transformation and progression. These studies also promise to uncover the molecular mechanisms of spontaneous regression and differentiation in neuroblastoma. This review will describe the most important genetic changes associated with neuroblastoma, and will outline the links between tumor genetics and tumor behavior.

PLOIDY

Evidence for the prognostic value of ploidy in neuroblastoma comes from flow cytometric and cytogenetic analyses. Flow cytometric analysis can easily be used to determine cellular DNA content. Although this analysis cannot detect specific chromosome rearrangements, such as deletions, translocations, or even gene amplification, it has been shown to correlate with biological behavior of neuroblastoma tumors, at least in a subset of patients. Using flow cytometric analysis, it was demonstrated that in infants, DNA content was significantly linked to tumor stage, with diploidy much more frequent in advanced tumor stages[12]. DNA content was also shown to discriminate between good and poor responders to chemotherapy. Hyperdiploidy is mainly observed in low stage tumors of younger patients with a favorable clinical outcome, whereas diploid tumors are associated with advanced tumor growth and significantly reduced survival probability[13-15].

Among 298 infants, hyperdiploidy, mostly in the near-triploid range, was closely associated with lower stages of disease and long-term survival, whereas diploidy was closely linked to advanced stages and treatment failure. In addition, in children 12-24 months of age, diploidy predicted early failure of chemotherapy, whereas half of the children with hyperdiploidy achieved long-term disease-free survival. Unfortunately, the DNA content lost its prognostic significance for patients over 2 years of age[16]. Cytogenetic analysis have been used to distinguish four ploidy levels in neuroblastoma tumors: near-diploid, near-triploid, near-tetraploid, and near-pentaploid tumors[17]. The near-diploid and near-tetraploid tumors were usually characterized by structural abnormalities, most commonly involving 1p, and by the frequent presence of amplified MYCN, and were mainly found in children older than one year. The near-triploid tumors were characterized by three almost complete haploid sets of chromosomes, with few structural abnormalities, and were predominantly found in infants with favorable outcome. Patients with near-pentaploid tumors, like those with near-triploid tumors, had favorable clinical and biological factors and high survival rates. In contrast, unfavorable prognostic factors were observed in patients who had either near-diploid or near-tetraploid tumors, and this subset of patients had a poor outcome[17-19]. The cytogentic approach may introduce sample bias as only a fraction of tumors yields karyotypes suitable for evaluation. Nevertheless, this observation indicates that discrimination only between diploid and aneuploid tumors may not be useful. Near-tetraploid tumors with poor prognosis are predominantly observed in older neuroblastoma patients with advanced disease, whereas near-triploid tumors are less frequent in this age group. This would explain why in early flow cytometry studies ploidy lost its prognostic power in older children[16]. Near-diploidy and near-tetraploidy have been identified as the most significant genetic marker for both poor event-free survival and poor overall survival in multivariate analysis[20].

More recently, comparative genome hybridization (CGH) has been extensively used for genome-wide screening of gains and losses in neuroblastoma[21-26]. With CGH, differentially labeled test and reference genomic DNA are co-hybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of DNA copy-number variation[27], albeit at limited (-20 Mb) mapping resolution. With respect to ploidy changes in neuroblastoma, CGH analyses have confirmed and extended the findings from flow cytometry and classical cytogenetic analysis: triploid tumors with favorable prognosis are mainly characterized by numerical chromosome imbalances with only few structural abnormalities. This includes the typical pattern of gains for chromosomes 1, 2, 6, 7, 8, 12, 13, 17, 18, and 22, and losses for chromosomes 3, 4, 9, 11, 14, and X[23]. The biological significance of these numerical chromosome changes is unknown. Most of the chromosomes that are underrepresented in low stage tumors also show partial loss in high stage tumors. Similarly, gain of chromosome 17, particularly 17q, is the most frequently observed chromosomal imbalances. Gain of the whole chromosome 17 is predominantly observed in low stage tumors, whereas partial gain of 17q is associated with high stage tumors[23].

To determine the prognostic value of ploidy and 1p deletion, karyotype and interphase 2-color FISH analyses using terminal 1p (D1Z2) and pericentromeric 1q (D1Z1) probes were performed in 246 neuroblastoma patients, including 186 patients detected by mass screening and 60 detected clinically[18]. They demonstrated that number of chromosome 1 correlated closely with the ploidy level, suggesting that tumors with disomy, trisomy, tetrasomy and pentasomy of chromosome 1 may be considered as tumors with diploidy, triploidy, tetraploidy and pentaploidy, respectively. Of tumors detected by mass screening, 90% were characterized by trisomy 1 with or without 1p deletion, and near-triploidy. The majority of tumors detected clinically with negative early mass screening result belonged to the disomy 1 group with or without 1p deletion, and near diploidy. Patients with disomy 1 and normal 1p had intermediate prognosis, while patients with disomy 1 and deleted 1p had poor prognosis; trisomy 1 with normal 1p and trisomy with deleted 1p patients both had favorable prognosis. This indicates that cytogenetic 1p deletion is a poor prognostic factor for diploid tumors, but not for triploid tumors, implying that structural rearrangement, such as 1p deletion, may have different prognostic impact dependent on tumor cell ploidy.

BIOLOGICAL SIGNIFICANCE OF PLOIDY

Many neuroblastomas have higher than normal ploidy states, sometimes in the pentaploid range. Aneuploid tumor cells in neuroblastoma might have emerged from tetraploidization and subsequent bipolar, tripolar, or tetrapolar divisions[28]. Supernumerary centrosomes leading to multipolar divisions have been implicated in both chromosome missegregation and the generation of aneuploid cells in various cancer types, including neuroblastoma[29]. A defect of spindle formation may cause incomplete segregation during mitosis. Thus, such a defect in a tetraploid cell undergoing a tripolar division could lead to one near-triploid and one near pentaploid cell. In fact, in neuroblastoma tumors with more than one tumor cell clone, near-pentaploid tumor cells are often observed together with near-triploid tumor cells. An attractive hypothesis that would explain how ploidy state of the tumor determines clinical heterogeneity of neuroblastoma was offered[28] (Figure 1). This hypothesis is based on the assumption that both favorable triploid and unfavorable diploid tumors arise through the same genetic event, which is suggested from observations in familial neuroblastomas[30, 31]. This initiating tumorigenic event would be a mutation in a classical tumor suppressor gene with recessive effect at cellular level[31, 32]. Tetraploidization and subsequent multipolar division of a diploid cell heterozygous for a mutation in such a gene would give rise to diploid and tetraploid daughter cells with no normal allele and highly malignant phenotype, or triploid daughter cells with at least one normal allele and less malignant phenotype (Figure 1). A candidate locus for this gene has been localized recently by genome-wide linkage analysis to chromosome band 16p12 in familial neuroblastomas[33], whereas other loci, such as chromosome 1p, have been excluded[34, 35]. Although it has been assumed that a familial neuroblastoma predisposition gene would function as a tumor suppressor, it is possible that dominant mutations may predispose to neuroblastoma.

ALLELIC LOSS OF 1p

Alterations of 1p characterize a wide range of human malignancies, including both solid tumors and hematological cancers[36]. In neuroblastoma, early cytogenetic analyses originally reported recurrent deletions of distal 1p (1p36) resulting in partial monosomy as the most frequent structural abnormality for both neuroblastoma tumors and cell lines[37-39]. Importantly, both constitutional and somatic deletions and translocations have been observed. A smallest region of overlapping deletion (SRO) was mapped cytogenetically to 1p34-p36[40], and it was suggested that the deletion results in the loss of a tumor suppressor locus (tumor suppressor gene; TSG) critical to the development of neuroblastoma. Functional support for a 1p tumor suppressor comes from the observation that the transfer of 1p chromosomal material into the neuroblastoma cell line NGP suppressed tumorigenicity and induced differentiation[41].

COMMON REGION OF DELETION AT 1p36.3
In a clinically and biologically representative study group approximately 35 percent of all neuroblastoma have 1p deletions of variable length[42-45]. A large number of molecular analyses in primary tumors has confirmed and refined the SRO, mainly detecting loss of heterozygosity (LOH) with polymorphic markers mapped to 1p[42, 46-49]. These efforts resulted in a SRO within 1p36 defined proximally by D1S244 and distally by D1S80. The low incidence of small interstitial deletions within 1p36 for long has made it difficult to further narrow the SRO, a prerequisite for positional cloning. Furthermore, although several 1p36 rearrangements have been identified in neuroblastoma cell lines, along with a constitutional translocation t(1;17)(p36.31-36.13;q11.2-12) in a patient with multifocal neuroblastoma[50], these chromosomal breakpoints are dispersed throughout a large genomic region. Homozygous deletions, which usually span relatively short genomic regions and have been instrumental in the identification of several TSGs, including RB1, WT1, and CDKN2A, were not found within 1p36 in a large survey[51].

Only recently the SRO was refined to a size of approximately 1 Mb within 1p36.3. One 1Mb SRO is defined by the region of LOH in a primary tumor that extends distally from D1S214, and by a constitutional deletion between D1S468 and D1S2826 in a patient with neuroblastoma[52]. Independently, a smallest candidate region of approximately 1 Mb was defined between the microsatellites D1S2731 and D1S2666 mapping to the chromosomal subband 1p36.3[53]. Both regions appear to overlap in the vicinity of marker D1S214. Neuroblastoma cell line NGP has a balanced translocation t(1;15)(36.2;q24), including a 2 Mb DNA duplication at 1p36.2[54]. Although the proximal breakpoint defined by the duplication appears to map outside the 1p36.3 SRO, the distal breakpoint, which maps to between D1S160 and D1S214, probably lies within the 1 Mb SRO. Several new genes mapping near this breakpoint region were identified recently and should be analyzed in more detail[55].

Recently, the first homozygous deletion spanning an approximately 500kb region at D1S244 has been identified in two neuroblastoma cell lines[56]. However, this homozygous deletion is localized proximal to the refined 1 Mb SRO, which would make a single tumor suppressor gene within 1p36.3 unlikely. A terminal 1p36 deletion syndrome has been described which is associated with mental retardation and craniofacial features[57, 58]. The prevalence of this deletion (1p36.3) is estimated to be 1 in 5000, making it the most common terminal deletion[58]. In all patients analyzed so far the deletion is distal to D1S228, in some the large deletions include the 1 Mb SRO within 1p36.3[59]. Two cases with terminal 1p36.3 deletion syndrome have been reported who developed neuroblastoma[52, 60], whereas none of the originally published cases has developed neuroblastoma[59]. This indicates that neuroblastoma is not a common feature of this syndrome. It remains unclear, whether some rare patients with 1p36.3 deletion syndrome may have a predisposition to neuroblastoma dependent on their deleted regions, or whether the two published cases are simply due to coincidence.

ONE OR MORE TSG LOCI IN 1p

Several observations suggest the existence of more than one commonly affected 1p locus in neuroblastoma. Patients with large 1p deletions have poorer outcome than patients with short or interstitial deletions[61]. While tumors with large 1p deletions were associated with adverse prognostic factors, such as diploidy or tetraploidy, and amplified MYCN, all tumors with small interstitial deletions were in the triploid range with a high proportion of tumors detected by mass screening. The existence of two distinct deleted regions was also suggested by LOH at polymorphic loci in clinically identified neuroblastomas [62, 63]. It appears that distinct loci are involved in neuroblastoma with and without MYCN amplification, since these tumors show different types of SRO[64]. Most MYCN-amplified neuroblastomas also have deletions of 1p, whereas MYCN single copy tumors show 1p LOH in only 15-20 percent of cases[42, 65]. 1p deletions of MYCN-amplified tumors are very large, always at least including a region from 1p35-1p36.1 to telomere. In contrast, in MYCN single copy cases, 1p deletions were described to be consistently smaller, and a commonly deleted region maps to 1p36.3. Thus, a second tumor suppressor locus inactivated by the 1p deletions in MYCN-amplified neuroblastomas has been postulated [63, 66]. This TSG was suggested to be localized at 1p35-36.1, just distal to the deletion border of the smallest 1p deletion found in MYCN-amplified cases[63, 67]. The smallest SRO of the MYCN single copy tumors is included into the larger SRO of MYCN-amplified tumors, implying that a distal suppressor locus in 1p36.2-3 must also be deleted in MYCN-amplified tumors.

IMPRINTING OF THE PUTATIVE 1p36 TSG

A non-random distribution with preferential deletion of the maternal allele in single copy MYCN, but not MYCN-amplified tumors was observed[65], which would imply that the distal TSG locus at 1p36.3 may be subject to genomic imprinting. This would indicate that of the two alleles of the putative tumor suppressor, one would be target of methylation or acetylation during spermatogenesis, without altering the genetic information encoded in the DNA itself, but with the consequence of reduced or even absent expression of this allele in the offspring. Somatic loss of the active maternal copy through LOH would then contribute to tumorigenesis[68]. Imprinting as an alternative mechanism of TSG inactivation involved in tumorigenesis has been described in various cancer types, e.g. Wilms tumor[68]. Currently, there is consensus that in neuroblastoma with MYCN amplification frequently characterized by a large 1p deletion region the parental origin of the deleted allele is of random distribution[69]. In contrast, the situation in MYCN single-copy tumors is less clear: whereas some reported preferential loss of the maternal allele[63, 65] others, albeit in a small number of patients, described random allelic loss[49, 70].

CLINICAL SIGNIFICANCE OF 1p ALTERATIONS

Early cytogenetic analyses in a small number of patients reported a correlation of 1p deletion with poor prognosis[19, 71]. As only a fraction of tumors yields karyotypes suitable for evaluation, the classical cytogenetic approach may introduce sample bias. PCR-based analyses of 1p36 status in primary neuroblastomas using polymorphic markers clearly documents an association of 1p36 LOH with adverse prognostic markers, such as age over 1 year at diagnosis, metastatic disease and MYCN amplification. Survival analyses, however, gave conflicting results[42, 48, 49, 72]. 1p LOH in combination with amplified MYCN was associated with decreased overall survival, whereas 1p LOH alone was not[73]. In other studies, however, LOH 1p has been identified as the most powerful predictor of poor outcome[66, 74]. Discrepancies were mainly due to small, heterogeneously assessed and treated populations, which did not allow detailed subset analyses. In addition, some studies analyzed event-free survival, whereas others measured overall survival. More recently, a large series (238 cases) analyzed under both criteria was presented. In this study, LOH 1p36 was a significant independent predictor of decreased event-free survival, but had no significant effect on overall survival in multivariate analysis[45]. In contrast, amplified MYCN was a more powerful prognostic factor for decreased overall survival. This implies, that determination of 1p36 allelic status may be useful for predicting which neuroblastoma patients with otherwise favorable clinical and biological features are more likely to have disease progression, but can be salvaged with additional therapy. Using two-color interphase FISH, the 1p36 status in a Japanese cohort with a substantial number of tumors from screened patients was determined[18]. As the number of chromosome 1 alleles correlates well with ploidy in the same tumor, two-color interphase FISH analyses using subterminal 1p (D1Z2) and pericentromeric 1q (D1Z1) probes can determine presence or absence of the 1p deletion, and also tumor cell ploidy in a single experiment. Event-free survival was lowest in the disomy 1 with 1p deletion group, intermediate in the disomy 1 with normal 1p group and highest in both trisomy 1 with normal 1p and trisomy 1 with 1p deletion groups. This indicates that the negative prognostic impact of 1p loss was restricted to diploid tumors, with loss of 1p from trisomy 1 tumors having no impact on survival. The majority of trisomy 1 tumors with 1p deletion were detected by mass screening. In a cohort mainly consisting of clinically detected neuroblastomas, 42 percent of 1p36 deletions were found in aneuploid tumor cells leading to allelic imbalance with at least 2 regular copies of chromosome 1 and additional 1p36 deleted copies[75]. In this study, interphase FISH revealed a strong intratumoral heterogeneity with more than one cell population especially in imbalance tumors which was not appearent in molecular assays. The majority of tumors with allelic imbalance revealed a tetrasomy 1 with two of the same 1p deleted chromosomes. Imbalance tumors were associated with adverse clinical and prognostic markers, and patient’s prognosis (EFS) was as poor as in disomy 1 tumors with 1p deletion. These tumors may have developed through tetraploidization of an 1p deleted disomic clone, or through tetraploidization of an disomic clone with normal 1p and subsequent deletion events. Taken together, the biological effect of 1p deletions has remained unclear. The genomic complexity of the region and the large size of the deletions hamper any overall synthesis of the involvement of genes localized in this region in neuroblastoma tumorigenesis. Several candidate tumor suppressor genes (TSGs) have been proposed, but none has been shown to contain tumor-specific mutations, indicating that alternate mechanisms of TSG inactivation, such as epigenetic mechanisms of gene inactivation or haploinsufficiency also have to be considered. Linkage analyses in familial neuroblastomas have excluded distal 1p to harbor the neuroblastoma predisposition gene[35]. The elucidation of the genomic structure and the informative complexity of this region is a prerequisite for determining the precise mechanism of altered activity of gene or genes localized in this region. In addition, structural alterations of chromosome 1 has to be evaluated together with coincident genetic changes. 1p alterations are variably detected together with other genetic alteration, such as amplified MYCN, 17q gain, diploidy/triploidy, and each combination appears to have divergent impact on tumor growth characteristics.

MYCN AMPLIFICATION

Amplified MYCN is one of the most prominent genomic abnormalities of neuroblastomas, and is prototypic for the significance of proto-oncogene amplification in tumorigenesis. It was originally detected by expression profiling of oncogenes in human neuroblastoma cells [76]. Because cytogenetically analyzed neuroblastoma cells contained conspicuous chromosomal abnormalities, homogeneously staining regions (HSRs) or double minutes (DMs) indicative of amplified DNA, it was suspected that the high expression seen in expression profiles was the consequence of gene amplification. This suspicion was verified by DNA analyses[76]. These original studies also established the amplification of MYCN in a neuroblastoma tumor in addition to cell lines. A subsequent study confirmed amplified MYCN in a substantial proportion of neuroblastomas [77].
DMs which predominante in primary tumors, and HSRs in cell lines were found as the chromosomal sites of amplified MYCN[78]. HSRs are generally located on different chromosomes, not at the resident site 2p24 of MYCN [78, 79]. Amplification values in neuroblastoma may range between 5-fold and more than 500-fold, usually values of around 50- to 100 fold are seen in tumors. Neuroblastoma cells lacking amplification are not necessarily single copy for MYCN. Instead, MYCN can be duplicated at 2p24, as shown by fluorescence in situ hybridization[80]. In addition to cell lines, duplication has been seen in primary tumors using FISH and CGH[22]. No systematic survey of tumors has yet been carried out, and therefore the frequency of duplications among tumors has remained unknown. The biological significance of duplication is emphasized by reports of neuroblastomas arising in children with constitutional duplication of 2p (p23-pter) including the MYCN locus[81, 82]. It is unclear whether duplication represents a prelude to amplification or an alternative pathway for activating the oncogenic potential of MYCN. MYCN copy number is usually consistent within a tumor, not only at different tumor sites, but also at different times in vivo[83]. This suggests that amplified MYCN, in positive tumors, is generally present at the time of diagnosis. FISH analyses of primary tumors, which allow detection at single cell level, have revealed that individual cells from MYCN amplified tumors typically stray widely from the mean copy numbers suggested by molecular analyses[84, 85]. The molecular pathways by which MYCN is amplified have remained enigmatic. The end-point of amplification appears to be a tandem arrangement of unit size amplicons in an HSR on a chromosomal position different form 2p24 with retention of the single copy MYCN at 2p24. These observations have suggested a model of the amplification process involving unscheduled replication and recombination to produce circular extrachromosomal elements, which could be visualized as a DM. At some point this extrachromosomal DNA could integrate into any chromosomal site and undergo several cycles of in situ amplification.

The complexity of amplified DNA encompassing MYCN can range from 100kb to more than 1 Mb [86]. A core 100- to 200 kb domain encompassing MYCN has been found consistently without rearrangements. In most HSRs several hundred kilobase DNA segments of unit size length without noticeable rearrangement compared with the normal genomic organization are present in an direct repeat head-to-tail tandem arrangement[86]. The large size of the amplified DNA in relation to the size of the MYCN gene raises the possibility that additional genes are coamplified. From several technological strategies to identify coamplified genes MYCN has emerged as the only consistently amplified gene, although in about 50-70% of the MYCN-positive cases the DDX1 gene, which maps within 400 kb 5’ of MYCN is found coamplified[87, 88]. More recently, neuroblastoma amplified gene (NAG) was also shown to be coamplified in 70% of MYCN- amplified neuroblastomas and to map telomeric to MYCN[89]. So far, no amplification of DDX1 or NAG without concomitant MYCN amplification has been noted, suggesting that MYCN is functionally responsible for the maintenance of the 2p24-amplified DNA.
These observations have raised the question, whether additional genes may be amplified in neuroblastoma, which are not restricted to the MYCN amplicon. In neuroblastoma cell lines, additional DMs or HSRS were identified not harboring MYCN. Using reverse genomic hybridization, amplified DNA was found to be derived from chromosome 12 band q13-14. Subsequent analyses showed 30-40-fold amplification of the MDM2 gene, abundantly expressed, both in some cell lines and a primary tumor, in addition to amplified MYCN[90]. This non-syntenic amplification of the MDM2 gene appears to be a rare event in neuroblastoma cells and has been seen exclusively in conjunction with amplified MYCN. More recently, co-amplification of the homeobox gene, MEIS1, which map to band 2p14, was described in the neuroblastoma cell line IMR-32[91]. Again, amplified MEIS1 was exclusively observed together with MYCN amplification. It is possible that neuroblastoma cells with several amplified genes have a particularly pronounced genomic instability, although it is not clear why amplification in neuroblastoma cells is restricted to certain genetic loci.

CLINICAL SIGNIFICANCE OF AMPLIFIED MYCN

Studies performed independently by various groups have shown a significant correlation between amplified MYCN and advanced stage disease; for example, in an early study, amplified MYCN was observed in 24 of 48 (50%) stage 3 and stage 4 tumors, but was not detected in 15 stage 1 and stage 2 tumors[77]. In the absence of amplified MYCN, overall survival was approximately 60% over a 5-year period, but only 10% of patients survived a 1-year period when MYCN was amplified at more than 10 copies[92]. Several studies confirmed this association with progressive disease and poor outcome[93-95]. Furthermore, a significant correlation between poor outcome and amplified MYCN was also observed when comparing patients over 1 year of age and with patients under 1 year[96]. In a multivariate analysis, amplified MYCN was identified as the only genetic factor which added significant prognostic information to clinical variables, such as age and stage[1]. Thus, amplified MYCN has been shown to characterize a subset of neuroblastoma with extremely aggressive growth potential. As such, it is established as a powerful clinical marker of high-risk low stage neuroblastoma. Currently, MYCN status is, so far, the only tumor genetic variable used as a basis for treatment stratification in neuroblastoma clinical trials.

MycN PROTEIN

High levels of MYCN expression are observed in MYCN-amplified tumors. DNA sequences have not revealed mutations in the MYCN coding sequence. In line with this, the biological activities of MycN from normal and from tumor cells have not been found to differ in experimental cell systems [97, 98]. It appears that it is the increased dose of a wild-type gene that contributes to tumorigenesis. Circumstantial evidence suggests that all copies of the amplified gene are transcriptionally active [99], which would explain the high level of MYCN mRNA in cells carrying amplified MYCN[100]. Obviously, most of this mRNA is translated, generating high levels of a 64 kDa nuclear phospoprotein [101], which forms a transcription complex by associating with a number of other nuclear proteins (for review see[102]). Experimental approaches have not demonstrated a functional difference between MycN and Myc proteins. Yet, of the two genes neuroblastomas amplify exclusively MycN, not Myc, and it is possible that MycN has an as yet unidentified role specific for neuronal cells. While amplified MYCN is a generally accepted prognostic parameter, there is conflicting experience regarding the potential prognostic significance of expression products, both mRNA and protein. Whereas one study demonstrated no significant correlation of MYCN expression and MycN protein levels with patients prognosis[103], another reported that in patients older than one year with MYCN non-amplified tumors, high levels of MYCN expression are associated with poor outcome[104].
The oncogenic properties of MYCN overexpression have been demonstrated in various experimental systems. Ectopic expression can assist mutationally activated RAS in the tumorigenic conversion of primary rat embryo cells, converts established cells of the rat and of humans to tumorigenecity in vitro[97, 105, 106]. Targeted expression in MYCN transgenic mice elicits development of neuroblastomas[107]. Mice homozygous for the transgene exhibited higher levels of MYCN expression than did the heterozygous mice, and demonstrated both an increase in incidence and a decrease in latency of tumor formation.
A number of reports emphasizes the role of MYCN, and the closely related MYC, in apoptosis. MYCN expression sensitizes neuroblastoma cells to enter programmed cell death (apoptosis) following exposure of cells to interferon-g or cytostatic drugs used in chemotherapy [108, 109], while Myc enhances the susceptibility to apoptosis in a variety of cells like fibroblasts and neurons (Evan, Wyllie et al. 1992, Wert, Kennedy et al 2001). Why then are many neuroblastomas resistant to chemotherapy despite enhanced expression levels of MYCN? One possibility is that apoptotic pathways are impaired. There is, in fact, precedence for this apoptotic dysfunction. For instance, absence of CD95, Apaf-1 or Caspase-9 confers oncogenic activity to overexpressed MYC[[110, 111]. Further, oncogenic cooperation between MYC and BCL2 appears to be result of an anti-apoptotic effect where BCL2 blocks MYC mediated apoptosis while proliferation-stimulation by MYC expression is retained[112]. More recently, CASP8 was shown to be epigenetically inactivated in MYCN-amplified neuroblastomas in vitro and in vivo[113].

17q GAIN

Early cytogenetic studies have first documented chromosome 17 abnormalities in neuroblastoma. In addition to chromosome 1p abnormalities, additional copies of 17q is a consistent finding in cell lines and primary tumors[114]. Functional evidence for a direct role of chromosome 17 material in the development of neuroblastoma comes from chromosome-transfer experiments: while transfer of 1p material induced neuronal differentiation in the cell line NGP, chromosome 17 material completely suppressed tumor-forming ability of neuroblastoma cells, suggesting that both chromosome 1p and 17 harbor a tumor suppressor gene[41]. The advent of FISH in the early 1990s has led to a more accurate description of complex rearrangements and unbalanced chromosome changes involving segments of unknown origin. Several studies of neuroblastoma cell lines and tumors have revealed a high frequency of unbalanced translocations involving chromosome 17q[115-119]. In addition to two or more normal chromosomes 17, a further segment of 17q12-qter can be translocated to numerous partner chromosomes, indicating that in addition to relative gain of 17q, genetic information on the partner chromosome is lost. More than 20 different chromosome regions, most frequently chromosome 1, followed by 11q, have been described to be involved in 17q translocations. More recently CGH analyses have substantially contributed to the picture of unbalanced 17q gain in primary neuroblastomas[21, 24, 120, 121].
Taken together, about 50% of neuroblastomas have an additional segment of 17q, therefore gain of 17q is the most frequent genetic alteration in neuroblastoma. Gain of 17q is more common at advanced stage, in tumors from children aged over one year, and in tumors showing 1p loss, MYCN amplification and ploidy in the diploid or tetraploid range. In contrast, triploidy with whole chromosome 17 gain is associated more often with neuroblastomas showing favorable clinical and tumor genetic features[122]. In the majority of cases amplified MYCN, 1p deletion and 17q gain coexist in the same tumors. Amplified MYCN rarely, if ever, occurs without either 1p deletion or 17q gain or both, implying that MYCN amplification is a later event in the sequence of genetic aberrations underlying neuroblastoma progression[122].

CLINICAL SIGNIFICANCE OF 17q GAIN

Initial studies had demonstrated that 17q gain was significantly associated with tumor progression [74, 123], even among patients without amplified MYCN or 1p deletion, indicating that detection of 17q gain might identify a larger proportion of high-risk tumors than other tumor genetic parameters [124, 125]. Furthermore, discriminative power of 17q was also reported in low-stage tumors (1, 2, 3 and 4s) [126]. Results from a collaborative compilation of data from six European centers with 313 cases identified 17q gain as the most powerful prognostic factor of survival in multivariate analysis with other clinical and tumor genetic parameters, including 1p deletion and MYCN amplification. In stepwise multivariate analysis, significant independent predictors of lethal outcome were 1p deletion (p=0.02), stage 4 disease (p=0.004), and 17q gain (p<0.001)[122]. In summary, these studies seem to suggest the independent prognostic importance of unbalanced gain of distal 17q in predicting patients at high risk for tumor progression and survival.

BIOLOGICAL SIGNIFICANCE OF 17q GAIN

Although the prognostic significance of 17q gain has been recently demonstrated, the underlying molecular mechanisms dictating this clinical course are completely unclear. The frequent occurrence of 17q translocations and the significant association with poor overall survival strongly suggest that the region of chromosome 17 gain includes a gene, or genes, critical for tumor progression. If so, there are in principle two possible ways in which such a gene could be affected by these rearrangements: either the unbalanced translocation disrupts a gene close to the breakpoint or the gain of 17q material could alter the gene dosage of one or more genes localized distal to the breakpoints. FISH analyses have indicated that the breakpoints were clustered in the proximal half of 17q from D17Z1 to MPO, with a shortest region of gain extending from MPO (17q23.1) to 17qter[115, 116]. At least seven different breakpoints were identified being randomly distributed within this region[119, 127]. The findings strongly favor a gene dosage effect, rather than rearrangement of a specific gene on chromosome 17q[118]. Early chromosome-transfer experiments had demonstrated that transfer of a whole chromosome 17 completely suppressed tumor-forming ability of neuroblastoma cell line NGP harboring an unbalanced t(6;17)(q27;q21)[41]. This implicates that a copy number imbalance between the segments proximal and distal to the 17q breakpoints being responsible for tumor-forming ability. Currently no data are available whether different breakpoints within chromosome 17q have divergent effects on patient’s prognosis, or not.
Several candidate genes have been proposed to be responsible for the 17q gain effect on tumor growth characteristics. In principle, each gene within the translocated 17q segment is a candidate, making the definitive proof for each candidate’s contribution almost impossible. Survivin, an anti-apoptosis protein, recently mapped to 17q25, is one candidate as its expression correlates strongly with adverse clinical factors, such as age and stage[128]. In addition, targeted expression of survivin was able to inhibit apoptosis induced by retinoic acid in a neuroblastoma cell line[129]. NM23-H1 at 17q21-22 is another candidate. Overexpression of nm23-H1 has been demonstrated in aggressive neuroblastoma tumors. Furthermore, a mutation (ser120-to-gly) has been identified in a subset of advanced stage neuroblastomas. One possibility could be that 17q gain specifically increases the expression of a mutant nm23-H1 protein with altered biological qualities contributing to tumor progression[130]. It can be assumed that the biological effect of 17 gain has a complex basis not only restricted to genes localized proximal or distal from the given breakpoints on chromosome 17. Each translocation of 17q material is associated with a deletion event on a partner chromosome, most frequently chromosome 1p, followed by 11q. Deleted chromosome regions may contribute substantially to tumor phenotype. Specific combinations of genetic changes, including 17q gain, 1p deletion and amplified MYCN have been used to identify at least two genetically distinct types of progressing tumors; the first with 17q gain but no amplified MYCN associated with frequent deletion of 11q or deletion of 1p, and the second with 17q gain, 1p deletion and amplified MYCN [131].

ALLELIC DELETION AT 11q

Cytogenetic analyses have reported 11q deletion in about 15% of neuroblastomas[132]. Functional evidence for a tumor suppressing effect of chromosome 11 was demonstrated by transfer of an intact chromosome 11 into the neuroblastoma cell line NGP inducing differentiation[41]. Constitutional rearrangements of 11q have been observed in some neuroblastoma patients, including a deletion of 11q23-qter, balanced translocations involving 11q21 and 11q22, and an inversion of 11q21-q23[133-135]. The role of these constitutional changes is not clear, but it has been speculated that disruption of one or more 11q genes may predispose to the development of neuroblastoma. LOH studies detected 11q loss in 5-32% of the tumors[44, 48, 136]. Loss of the whole chromosome 11 was observed in 19% while unbalanced 11q LOH in 22% of primary neuroblastomas[137]. Loss of the whole chromosome 11 was strongly associated with low stage tumors whereas unbalanced deletion of 11q was predominantly observed in high stage tumors without amplified MYCN[137, 138].
A number of CGH studies have confirmed the unbalanced 11q deletion in approximately 20% of primary neuroblastomas, and loss of whole chromosome 11 was a frequent finding in near triploid low stage neuroblastomas[21, 22, 24, 121]. Unbalanced 11q deletion was present in more than 50% of stage 4 neuroblastomas without MYCN amplification[22]. This genetic subgroup is also characterized by a positive correlation with deletion events, such as losses of 3p, 4p, and 14q, and an inverse correlation with 1p deletion, and furthermore 17q gain was consistently present[22, 23]. This indicates that unbalanced 11q deletions are a frequent event during the malignant evolution of MYCN non-amplified tumors and define a distinct genetic subgroup of stage 4 tumors. Loss of 11q was significantly associated with adverse clinical parameters, such as age over 1 year, stage 4 disease and unfavorable histology[139]. However, in a multivariate analysis unbalanced 11q deletion did not add information in addition to age and stage[139].
Deletion events affecting 11q are predominantly large and terminal. A single region of 2.1 cM within 11q23.3, flanked by markers D11S1340 and D11S1299, was deleted in all tumors with 11q LOH[138]. Deletions of 11q often have been seen in concert with gain of 17q. FISH analyses have demonstrated that, after 1p, chromosome arm 11q is the second most common partner for 17q translocations[116]. Such translocations, resulting in concurrent loss of distal 11q and gain of 17q, account for approximately half of the 11q deletion[23]. This further emphasizes the link between frequently observed deletion and gain of 17q material in neuroblastoma. Therefore, LOH studies assessing the prognostic value of particular losses should take into account the 17q status of each individual tumor.

EPIGENETIC ALTERATIONS

In addition to amplified MYCN, 1p deletion, 17q gain, 11q deletions, and ploidy changes, further non-random genetic alterations in neuroblastomas exist (table 1). In general, deletions are more common than defined non-random chromosomal gains in neuroblastoma. Molecular genetic characterization of deleted regions in neuroblastoma was largely influenced by the two-mutation hypothesis[31], predicting that LOH events are the second step in the inactivation of both alleles of a TSG. LOH analyses have identified a large number of regions of allelic loss, and genetically mapping of LOH events on a case-by-case basis was used to delineate a common region containing one or more putative tumor suppressor genes. The number of candidate genes that has emerged from LOH studies is small, with none meeting the criterion in the classical sense of a tumor suppressor gene. There are several possible explanations: (1) cytogenetic karyotypes, especially from advanced stage neuroblastomas, frequently include marker chromosomes and unidentified products of unbalanced translocations. It is conceivable that this karyotype instability increases the rate of LOH, and it seems possible, that not all of these LOH events are critical for tumor progression; (2) LOH could result in haploinsufficiency and the reduced activity of a particular gene at a given locus, alone or in combination with other genes, could contribute to tumorigenesis; (3) epigenetic mechanisms could play a role. In particular, there is growing evidence that silencing rather than mutation is a common mechanism for loss of suppressor gene function.
One of the strongest candidate tumor suppressor genes in neuroblastoma has emerged from an expression survey of apoptosis-related genes in neuroblastoma cell lines. A low or even absent expression of caspase 8, a cysteine protease involved in death-receptor induced apoptosis, was detected in a panel of neuroblastoma cell lines[113]. In neuroblastoma cell lines, the CASP8 gene was deleted or silenced by methylation while in neuroblastoma tumor samples, methylation of its promoter region was the predominant mechanism for its inactivation. In this study, complete inactivation of CASP8 occurred almost exclusively in neuroblastomas with amplified MYCN. CASP8 has been mapped to 2q33, a region of LOH in neuroblastoma detected by mass screening[44]. In contrast, CASP10, which is about 40 kb 5’ of CASP8, was not deleted or altered, indicating that alterations selectively affect caspase 8 expression[113, 140]. Inactivating point mutations in the CASP8 gene in neuroblastomas are rare, indicating that inactivation mainly occurs through deletion and methylation.
Further evidence for epigenetic gene inactivation in neuroblastoma comes from studies at the non-random deleted region on chromosome 3 originally detected by representational difference analysis (RDA)[141]. A SRO encompassing chromosome bands 3p25.3-p14.3 with 46 cM was defined[142]. Recently, a novel 3.p21.3 candidate tumor suppressor, a RAS association domain family protein (RASSF1A), was identified within the SRO[143], and has been shown to be epigenetically silenced by promoter methylation in primary neuroblastomas and other cancer types[143, 144]. It was postulated that silencing of RASSF1A contributes to aberrations of RAS signal pathways observed in neuroblastomas[145].
In addition, a significant correlation between RASSF1A and CASP8 methylation in neuroblastoma could be demonstrated[144], suggesting that a subset of neuroblastoma may have a CpG island methylator phenotype (CIMP) as described in colorectal cancer[146].

PERSPECTIVES

Neuroblastoma, despite of many advances in understanding its biological diversity and developmental molecular pathways has remained a dreadful disease of young children. At the same time, the fascinating multiplicity of clinical and biological phenotypes has attracted the attention of a growing number of clinical and basic scientists. It can be expected that their combined efforts inevitably will lead to an understanding of the molecular pathways governing both progression and spontaneous regression of neuroblastoma. This knowledge eventually should provide the platform from which new diagnostic tools can be developed and from which the design of patient-oriented and new types of therapies can be attempted.

Acknowledgements
Work of the authors is supported by Deutsche Forschungsgemeinschaft (Fellowship to F. W.; WE 2517/1) and by Deutsche Krebshilfe.

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Figure 2.

Figure 3.

Table 1.

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