Histopathologic Classification

In 1984, Shimada and colleagues first developed an age-linked classification system of neuroblastic tumors based on tumor morphology in which neuroblastomas were divided into two prognostic subgroups, favorable histology and unfavorable histology.¹³ In 1999, the International Neuroblastoma Pathology Classification (INPC) was devised, and then modified in 2003, and is an adaptation of the original Shimada system.^14,15 The INPC is based mainly on morphologic changes associated with the maturational sequence of neuroblastic tumors. It remains an age-linked classification that depends on the differentiation grade of the neuroblasts, the cellular turnover index (mitosis-karyorrhexis index [MKI]), and the presence or absence of Schwannian stroma. The INPC classifies neuroblastic tumors into three morphologic categories: neuroblastoma, ganglioneuroblastoma, and ganglioneuroma (Fig. 66-1).

Neuroblastomas are, by definition, Schwannian stroma poor (<50% of the tumor tissue) and can be subtyped as undifferentiated, poorly differentiated, or differentiating. Undifferentiated requires supplemental diagnostic methods such as immunohistochemistry, electron microscopy, or cytogenetics. Moreover, neuropil is not present. In poorly differentiated, <5% of tumor cells have features of differentiation, and neuropil is present. Differentiating tumors demonstrate >5% of tumor cells differentiating toward ganglion cells. To classify a cell as a differentiating neuroblast, there must be synchronous differentiation of the nucleus and eosinophilic cytoplasm.¹⁴

Additional factors that contribute to the prognostic distinction of stroma-poor neuroblastic tumors (neuroblastoma) as favorable or unfavorable subtypes include the MKI, which is defined as the number of tumor cells in mitosis or karyorrhexis per 5000 neuroblastic cells (i.e., low MKI, <100 cells; intermediate, 100–200 cells; high, >200 cells), and the patient’s age (<1.5 years, 1.5-5 years, >5 years) (Table 66-1). It has been hypothesized that neuroblastic cells with maturational potential require a latent period before demonstrating histologic evidence of differentiation. Therefore, there is a certain allowance for mitotic and karyorrhectic activities of neuroblastic cells in tumors in infants and younger children.¹⁶

The importance of this histopathologic classification was confirmed in a large, retrospective analysis reported by Shimada et al.¹⁷ The INPC classification of tumor histology provided independent prognostic information where tumors of favorable histology had a 90.8% probability of 5-year event-free survival [EFS] compared to 31.2% EFS for tumors of unfavorable histology. More recently, the INPC classification has been shown to add independent prognostic information beyond the prognostic contribution of age.¹⁸ Therefore, histopathology remains in the current multifactorial risk stratification for patients with neuroblastoma. This determination is particularly important in patients with MYCN non-amplified tumors who are older than 18 months and have stage 3 disease, or 12 to 18 months of age and have stage 4 disease, or have 4S disease. As the histopathologic pattern within a tumor can be heterogeneous, it is recommended that representative sections from at least 1 cm³ of viable, non-necrotic tissue be analyzed to determine histopathologic classification. The prognostic value of assessing the histopathology of a neuroblastoma after chemotherapy or radiation therapy has not been validated.

Stroma-rich neuroblastic tumors are classified as either ganglioneuroblastomas or ganglioneuromas. Ganglioneuroblastomas contain cells that are transitioning toward differentiation but are not completely differentiated/mature. Also, <50% of the total volume is made up of neuroblastic cells. Ganglioneuroblastomas can be further divided into ‘intermixed’ and ‘nodular’ subtypes, depending on the distribution of the neuroblastic cells. The distinction is important because of the significantly worse prognosis associated with the latter subtype, in which the neuroblastic clones that comprise grossly distinct nodules appear to be responsible for the aggressive phenotype for this subtype.¹⁶ Ganglioneuromas contain either maturing or mature cells, and lack any neuroblastomatous component. Most stroma-rich tumors (ganglioneuroblastoma, intermixed and ganglioneuroma, maturing subtype) are classified as ‘favorable’ by the INPC. However, the pathologic/prognostic classification of the ganglioneuroblastoma, nodular subtype is based on the morphologic evaluation of the neuroblastomatous nodule(s), and can, therefore, be unfavorable. Tumors that fit the criteria for ganglioneuroma, mature subtype, with abundant Schwannian stroma and fully mature ganglion cells, in the absence of neuroblasts, are considered benign, and are generally not considered for enrolment in protocols for neuroblastic tumors. Despite this, ganglioneuromas can be quite large and infiltrative, and attempts at removal can be associated with significant complications. In addition, survival does not seem to be influenced by extent of resection.¹⁹ Therefore, aggressive attempts at resection of ganglioneuromas are not recommended.

DNA Content

Normal human cells contain two copies of each of 23 chromosomes; thus, a normal diploid cell has 46 chromosomes. The majority (55%) of primary neuroblastomas are triploid or ‘near-triploid/hyperdiploid’ and contain between 58 and 80 chromosomes; the remainder (45%) are either ‘near-diploid’ (35–57 chromosomes) or ‘near-tetraploid’ (81–103 chromosomes).²⁰ The ‘DNA index’ of a tumor is the ratio of the number of chromosomes present to a diploid number of chromosome (i.e., 46). Therefore, diploid cells have a DNA index of 1.0, whereas near-triploid cells have a DNA index ranging from 1.26 to 1.76. Neuroblastomas that are near-diploid or near-tetraploid usually have structural genetic abnormalities, most frequently chromosome 1p deletion and MYCN amplification. Near-triploid or hyperdiploid tumors are characterized by almost three complete haploid sets of chromosomes with few structural abnormalities. Importantly, patients with near-triploid tumors typically have favorable clinical and biologic prognostic factors and excellent survival rates, as compared with those patients who have near-diploid or near-tetraploid tumors.²¹ This association is most important for infants with advanced disease as the prognostic significance of tumor ploidy appears to be lost in patients older than 2 years.²² Currently, ploidy only potentially impacts the risk group assessment of infants age 12–18 months with metastatic disease and infants with 4S disease in the COG risk stratification schema.

Amplification of MYCN

Investigation of the molecular biology of neuroblastoma began with the cytogenetic characterization of tumor-derived cell lines. These studies showed the frequent presence of extrachromosomal double-minute chromatin bodies (DMs) and chromosomally integrated homogeneously staining regions (HSRs) characteristic of gene amplification (Fig. 66-2).²³ Since that time, it has been shown that the amplified region was derived from the distal short arm of chromosome 2 (2p24) and contained the MYCN proto-oncogene. MYCN encodes a 64 kDa nuclear phosphoprotein that forms a transcriptional complex by associating with other nuclear proteins expressed in the developing nervous system and other tissues.²⁴ Enforced expression of MYCN increases the rates of DNA synthesis and cell proliferation, and shortens the G₁ phase of the cell cycle.²⁵ MYCN can also function as a classic dominant oncogene that cooperates with activated ras to transform normal cells.²⁶ Targeted expression of MYCN in transgenic mice results in the development of neuroblastomas.²⁷ This activity is potentiated when combined with mutations in ALK.²⁸

Overall, approximately 25% of primary neuroblastomas in children have MYCN amplification, with MYCN amplification being present in 40% with advanced disease but only 5–10% with low-stage disease.²⁹ The copy number, which can range from 5- to 500-fold amplification, is usually consistent among primary and metastatic sites and at different times during tumor evolution and treatment.³⁰ This finding suggests that MYCN amplification is an early event in the pathogenesis of neuroblastoma. Amplification of MYCN is associated with advanced stages of disease, rapid tumor progression, and poor outcome; therefore, it is a powerful prognostic indicator of tumor behavior.^29,31 Amplification can be detected either by routine metaphase cytogenetics or fluorescent in situ hybridization (FISH), and current therapeutic neuroblastoma protocols have incorporated the presence or absence of MYCN amplification into their risk stratification schema.

Chromosomal Changes

Also noted on early karyotype analyses of neuroblastoma-derived cell lines were frequent deletions of the short arm of chromosome 1.³² Deletions of genetic material in tumors suggest the presence (and subsequent loss) of a tumor suppressor gene. Although no individual tumor suppressor gene has been confirmed on chromosome 1p, recent data have identified CHD5 as a strong candidate for the tumor suppressor gene that is deleted from 1p36.31 in neuroblastoma.³³ Functional confirmation of the presence of a 1p tumor suppressor gene came from the demonstration that transfection of chromosome 1p into a neuroblastoma cell line resulted in morphologic changes and ultimately cell senescence.³⁴ Approximately 20–35% of primary neuroblastomas exhibit 1p deletion, as determined by FISH, with the smallest common region of loss located within region 1p36.³⁵ About 70% of advanced-stage neuroblastomas have 1p deletions.³⁶ Molecular studies have shown that there is a strong correlation between 1p deletion and MYCN amplification and other high-risk features such as age older than 1 year and advanced-stage disease.³⁵ One recent study has demonstrated that 1p deletions are independently associated with a worse outcome in patients with neuroblastoma.³⁷

Deletion of the long arm of chromosome 11 (11q) also appears to be common in neuroblastoma, being present in about 40% of cases. Unbalanced deletion of 11q (loss with either retention or gain of 11p material) is inversely related to MYCN amplification,37,38 yet is strongly associated with other high-risk features. Recently, Attiyeh et al., on behalf of COG, showed in a large cohort of patients that unbalanced deletion of 11q and 1p36 were independently associated with a worse outcome in patients with neuroblastoma.³⁹ Therefore, the duration of treatment for children with intermediate-risk neuroblastoma on the recent COG study was based, in part, on the 1p and 11q allelic status of the tumor.

Mutations

An example of proto-oncogene activation by point mutation involves the tyrosine kinase receptor, ALK, on the short arm of chromosome 2 (2p23). Receptor tyrosine kinases (RTK) are high-affinity cell surface receptors for many growth factors, cytokines, and hormones. When activated through ligand binding, these proteins mediate phosphorylation of tyrosine on target molecules or substrates, resulting in intracellular signaling and, ultimately, the regulation of normal cellular processes. Mutation of RTK’s can lead to constitutive activation of the signaling pathway in the absence of ligand. Recently, activating mutations of ALK have been shown to be the germline abnormality associated with hereditary neuroblastoma.⁴⁰ These mutations can also be somatically acquired, as can amplification of the gene, although the prevalence of ALK activation in sporadic neuroblastoma is not known.⁴¹ Activated ALK has proven to be a targetable abnormality in neuroblastoma, with drugs such as crizotinib, an anti-ALK antibody, showing efficacy.⁴² Further studies have identified loss-of-function mutations in the homeobox gene PHOXB2 on 4p13 that are also associated with familial neuroblastoma, particularly when occurring together with Hirschsprung disease and/or central hypoventilation.⁴³

Recently, inactivating mutations of ATRX, a transcriptional regulator that is part of a multiprotein complex that plays a role in regulating chromatin remodeling, nucleosome assembly, and telomere maintenance, have been found in neuroblastoma, particularly high-stage tumors in older patients.⁴⁴ ATRX mutations appear to be loss-of-function mutations associated with an absence of the ATRX protein in the nucleus, and with long telomeres. How these alterations lead to lengthened telomeres is uncertain. These results may provide a molecular marker and potential therapeutic target for neuroblastoma among adolescents and young adults. It may also delineate the subset of children with neuroblastoma who have a chronic but progressive clinical course when receiving standard therapeutic approaches and who may benefit from a different treatment strategy.

Other Molecular Abnormalities

Neurotrophins and their tyrosine kinase receptors are important in the development of the sympathetic nervous system and have been implicated in the pathogenesis of neuroblastoma. Three receptor-ligand pairs have been identified: TrkA, the primary receptor for NGF; TrkB, the primary receptor of brain-derived neurotrophic factor (BDNF); and TrkC, the receptor for neurotrophin-3 (NT-3).⁴⁵ TrkA appears to mediate differentiation of developing neurons or neuroblastoma in the presence of NGF ligand, and apoptosis in the absence of NGF.⁴⁶ High TrkA expression is associated with favorable tumor biology and good outcome⁴⁷ and is inversely correlated with MYCN amplification.⁴⁸ Conversely, the TrkB/BDNF pathway appears to promote neuroblastoma survival through autocrine or paracrine signaling, especially in MYCN-amplified tumors.⁴⁹ TrkB is expressed in about 40% of neuroblastomas, usually advanced-stage disease. TrkC is expressed in approximately 25% of neuroblastomas and is strongly associated with TrkA expression.⁵⁰

Other molecular abnormalities frequently detected in neuroblastoma include inactivation of caspase 8, expression of CD44, and overexpression of multidrug resistance genes. Studies have demonstrated inactivation of caspase 8, a component of the Fas death-signaling complex, in MYCN-amplified neuroblastomas.⁵¹ It has been proposed that inactivation of caspase 8 renders tumor cells resistant to apoptotic signals. CD44 is a cell surface glycoprotein that appears to play a role in tumor cell adhesion.⁵² In neuroblastomas, CD44 expression is inversely correlated with MYCN amplification and is undetectable in most disseminated neuroblastomas.⁵³ Multidrug resistance-associated protein (MRP) is an efflux pump whose expression in neuroblastoma appears to be correlated with MYCN amplification and poor prognosis.^54,55 The presence of MRP may explain the common clinical situation in which neuroblastomas initially respond well to chemotherapy but subsequently become resistant.

Genome-Wide Association Studies

Recently, microarray technologies have generated extensive amounts of data that have aided in identifying genomic (DNA) and transcriptomic (RNA) abnormalities associated with neuroblastoma. In addition, these abnormalities have been shown to have significant predictive power when anticipating outcome for these patients.56,57 Many of these findings were generated by large scale genome-wide association studies (GWAS). This is a technique whereby all or most of the genes of patients with neuroblastoma are analyzed to find differences with the population as a whole, looking for variations that are associated with the development and aggressiveness of neuroblastoma. The causal relationship between the DNA variant associated with the cancer is uncertain but an excessive inheritance of risk variants has been postulated to increase susceptibility to the disease. Several GWAS studies have been performed in patients with neuroblastoma and have identified a number of such genetic risk variants.⁵⁸ These observations suggest that developmental childhood cancers are likely influenced by common DNA variations, leading to the development of a putative genetic model.⁵⁸ Recent data suggest that the higher prevalence of high-risk disease in Black and Native American patients with neuroblastoma may be associated with certain genetic variants found more commonly in these ethnic groups.⁵⁹

One such type of variation is a single-nucleotide polymorphism (SNP) in which there is a variation in the DNA sequence for a single nucleotide between children with neuroblastoma and those without neuroblastoma, and with varying degrees of tumor phenotype. Another type of variation is copy number variation (CNV) which is an alteration of the DNA resulting in an abnormal number of copies of one or more sections of the DNA. CNVs correspond to relatively large regions of the genome that have been deleted or duplicated.

One method for detecting CNVs is by comparative genomic hybridization (CGH). Early CGH studies showed that gain of genetic material on the long arm of chromosome 17 (17q) is perhaps the most common genetic abnormality in neuroblastomas, occurring in approximately 75% of primary tumors.⁶⁰ It is unclear at this time how extra copies of 17q contribute to the malignant phenotype of neuroblastoma and which gene(s) on 17q are the critical ones. Nevertheless, gain of chromosome 17q is strongly associated with other known prognostic factors, but it may also be a powerful predictor of adverse outcome.⁶¹ More recently, GWAS studies have shown that inherited CNV at chromosome 1q21.1 is associated with neuroblastoma, implicating a neuroblastoma breakpoint family gene in early neuroblastoma genesis.⁶²

Other studies have revealed that common genetic variation at chromosome bands 6p22⁶³ and 2q35⁶⁴ are associated with susceptibility to high-risk neuroblastoma, providing the first evidence that childhood cancers can also arise from complex interactions of polymorphic variants. More recently, a GWAS study has identified common polymorphisms including germline SNP risk alleles and somatic copy number gain, resulting in increased expression of the cysteine-rich transcriptional regulator LIM domain only 1 (LMO1) at 11p15.4. These have been shown to be strongly associated with susceptibility to developing neuroblastoma, and often are associated with advanced disease and poor survival.⁶⁵

Finally, whole-genome sequencing of tumors, made possible recently by significant advances in technology, has been performed to investigate the genetic landscape of a variety of pediatric tumors.⁶⁶ Initially it was felt that early alterations of genes such as MYCN may underlie the rapid acquisition of cooperating mutations in key cancer pathways through chromosome instability. However, few recurring amino acid changes have been detected in neuroblastoma specimens, suggesting that the tumor genomes were more stable than previously believed.^44,67 Also, unlike the genetic landscape, the epigenetic profiles showed profound changes which suggests that epigenetic changes may have a more dominant role in pediatric tumorigenesis.

Laboratory Findings

Diagnostic Imaging

Bone Marrow Examination