Embryology

Neuroblastoma arises during development from neural crest cells committed to the sympathoadrenal lineage. The two predominant cell types comprising neuroblastoma are neuroblasts and Schwann cells. Neuroblasts derive from pluripotent cells that arise in the neural crest and eventually form various components of the sympathetic nervous system: the sympathetic ganglia, the chromaffin cells of the adrenal medulla, and the paraganglia, which are typical sites where neuroblastic tumors can arise. What causes persistence of these embryonal cells that develop into peripheral neuroblastic tumors is unclear. Genes that dictate neural development could be altered, causing defects in the normal differentiation and programmed cell death pathways, leading to uncontrolled proliferation and aberrant cell survival.

Microscopic neuroblastic nodules have been reported in fetal adrenal glands during development, peaking at around 17 to 20 weeks’ gestation and regressing by birth or within the first few months of postnatal life. Beckwith and Perrin (1963) termed these nodules, which were histologically identical to neuroblastoma, “neuroblastoma in situ,” which they noted during postmortem examinations of infants younger than 3 months of age who died of other causes. These lesions were detected with a frequency fiftyfold higher than the expected incidence of primary adrenal neuroblastoma and were initially thought to be neuroblastomas that regressed spontaneously. However, with new insights into the development of the adrenal gland, it is clear that these are remnants from normal fetal development. Indeed, the genetic profiles of normal developing neuroblasts and malignant neuroblastomas are similar in many respects, as shown by messenger ribonucleic acid (mRNA) expression profiling of sympathetic neuroblasts from human fetal adrenal glands.

Development of the Sympathetic Nervous System

The sympathetic nervous system is derived from the neural crest, a multipotent embryonic structure that appears early in development and is made up of a transient population of cells that migrate from the neural tube to the region of the dorsal aorta during development. These cells develop into sympathetic, parasympathetic, enteric, or sensory neurons governed by transcriptional regulators and growth factors as they exit from the neural tube, during migration or at sites where these cells differentiate. Development of the sympathetic phenotype is summarized in Figure 54-1 . Premigratory neural crest cells are specified by the expression of several markers, including FoxD3 and SOX10. Foxd3 is required for sublineage fate specification, migration, and survival. Zebrafish homozygous for foxd3 inactivating mutations have nearly a complete loss of sympathetic neurons and their precursor populations. Sox10 is responsible for the multipotency of neural crest–derived cells and is required for sympathetic nervous system and melanocyte development. At the dorsal aorta, prespecified SOX10 -positive sympathetic progenitors that are under the influence of extrinsic factors, bone morphogenetic proteins, start the process of differentiation into noradrenergic neurons. The basic helix-loop-helix (bHLH) transcription factor Ascl1 and the homeodomain transcription factor Phox2b are the first transcription factors that are expressed upon initiation of differentiation of sympathoadrenal precursors. Phox2a , a homolog of PHOX2B , is expressed downstream of both Phoxb and Ascl1, but its role in the initial stages of the sympathetic lineage remains undefined. Further neuronal differentiation occurs under the influence of the transcription factors Hand2 , Gata2/3, and Tfap2a , which interact in a complex regulatory network to ultimately induce the expression of tyrosine hydroxylase (TH) and dopamine beta hydroxylase. These two enzymes are required for catecholamine production and serve as markers of terminal sympathetic neuronal differentiation. Hand2 appears to have more of a role in noradrenergic cell proliferation than differentiation. A massive reduction in sympathetic ganglion size occurs after elimination of Hand2 in the neural crest of model organisms because of a nearly complete block in proliferation of immature sympathetic neurons. Thus different members of the gene regulatory network appear to have selective functions, some in proliferation and others in differentiation.

The development of the autonomic nervous system.

A series of growth factors and transcriptional regulators affect different stages of neurogenesis of neural crest–derived progenitor cells. Neural crest cells segregated from the neuroepithelium can be identified by the expression of forkhead box D3 (FoxD3) and sex-determining region Y box–10 (SOX10), among other markers. Progenitor cells differentiate into sympathetic, parasympathetic, enteric, or sensory neurons that are in part dependent upon instructive signals encountered early at or near the time of egress from the neural tube. Additionally, extrinsic cues encountered during migration or at sites where neural crest–derived cells differentiate influence patterns of gene expression.

(Modified from Howard MJ. Mechanisms and perspectives on differentiation of autonomic neurons. Dev Biol 277:271–286, 2005.)

Neural crest cells from the cranial region migrate from the neural folds into the pharyngeal arches and heart, where they play essential roles in septation of the single outflow of the heart (the truncus arteriosus) and in the formation of the conotruncal portion of the ventricular septum. It follows that coexistence of neuroblastoma with anomalies of other neural crest–derived structures is biologically plausible because of the shared roles of regulatory and developmental genes. For example, ablation of the cardiac neural crest results in congenital cardiovascular malformations in the chick embryo. Indeed, a significantly increased incidence of congenital cardiac malformations, especially those that involve neural crest–derived tissues, was noted in patients with neuroblastoma compared with other malignancies, such as leukemia. These cardiac anomalies were seen primarily in patients with neuroblastoma who were younger than 1 year and had lower stage tumors. Menegaux et al. (2005) confirmed the association of neuroblastoma with conotruncal cardiac anomalies, as well as with various urogenital anomalies; other studies have shown varying results. Although the malignant transformation of neuroblasts and the disruption of normal cardiogenesis are likely to be multistep processes, it is possible that shared initiating genetic lesions occur very early in development, causing derangement of common developmental pathways.

Genetic Disorders Associated with Neuroblastoma

Neuroblastoma may occur rarely in association with chromosomal anomalies. In addition to the increased incidence of neuroblastoma in girls with Turner syndrome, neuroblastoma has also been noted to occur in patients with Noonan syndrome. The gene encoding the protein phosphatase PTPN11 is mutated in 50% of patients with Noonan syndrome and is associated with an increased risk of leukemia and neuroblastoma. Other neuro-cardio-facial syndromes such as Costello syndrome have been associated with neuroblastic tumors. Mutations in the neurofibromatosis type 1 (NF1) gene have been detected in neuroblastoma cell lines, and the literature includes one case report of a patient with a germline NF1 mutation and neuroblastoma who had a homozygous deletion of the NF1 gene in the tumor. However, germline inactivation of Nf1 does not predispose to neuroblastoma in the mouse.

Persons affected with overgrowth disorders such as Beckwith-Weidemann syndrome are at risk for embryonal tumors, including neuroblastoma. The increased risk is mainly during the first 4 years of life, and current recommendations include quarterly evaluation with abdominal ultrasound until age 8 years. The molecular aberrations that predispose to neuroblastoma in particular are unknown, although maternal differentially methylated region 2 (DMR2) loss of methylation that occurs in 50% to 60% of cases is thought to confer a 1% to 5% risk of developing non–Wilm tumors, including neuroblastoma, hepatoblastoma, rhabdomyosarcoma, adrenocortical tumors, gonadoblastoma, and thyroid carcinoma. Mutations of the CDKN1C tumor suppressor gene (encoding the cyclin-dependent kinase inhibitor p57 ^KIP2 ), which occur in 5% to 10% of sporadic and 30% to 50% of familial cases, is associated with a 4% estimated risk of non–Wilm tumor formation. The X-linked Simpson-Golabi-Behmel syndrome type 1 (SGBS1), associated with mutations in glypican 3 and 4 (GPC3 and GPC4) genes, is characterized by prenatal and postnatal macrosomia, distinctive craniofacies with palatal anomalies, congenital heart defects, and neuroblastoma (reviewed in reference ). Surveillance is recommended for embryonal tumors starting in the newborn period.

Neuroblastoma Stem Cells

The natural course of high-risk neuroblastoma, which results in relapse and a refractory state after an initial good response, suggests the likely existence of cancer stem cells (CSCs) in these tumors. CSCs are a subpopulation of cells within a tumor with the ability to self-renew, generate differentiated progeny, and reproduce a heterogeneous cancer population. These cells have enhanced tumorigenicity, resistance to chemotherapy, and the ability to establish metastatic foci. Hansford et al. (2007) demonstrated that dissociated cells from neuroblastoma tumors and bone marrow metastases grow as spheres and are capable of self-renewal. Spheres generated from the tumors underwent differentiation under neurogenic conditions to form neurons. Neuroblastoma stem cells from high-risk tumors expressed markers of neural crest stem cells and clinical markers of neuroblastoma, exhibited a higher self-renewal rate than those from low-risk tumors, and formed metastatic tumors that could be serially passaged in xenograft models. Compounds that selectively targeted these tumor-initiating cells (TICs)—DEC14 and rapamycin—were identified; they induced regression of TIC tumor models in vivo and markedly decreased self-renewal or tumor initiation capacity. Moreover, compounds that targeted PI3K/Akt, PKC, aurora kinase, ErbB2, Trk, and polo-like kinase–1 (PLK1) emerged from a small molecule inhibitor library screen designed to identify signaling pathways important for the survival and self-renewal of neuroblastoma TICs. Genetic and chemical inhibition of PLK1 caused regression of TIC tumor models both singly and in combination with irinotecan, suggesting that this kinase may regulate TIC growth and survival.

Cluster of differentiation (CD)–133, in addition to being a marker of hematopoietic stem cells, is also a marker of TICs in several human cancers. CD133-positive subpopulations of cells with tumor-initiating and metastatic properties have been identified in neuroblastoma tumors. Similarly, the stemness-related polycomb repressive complex–1 (PRC1) protein BMI1 is thought to have a role in tumor sphere formation. BMI1 is thought to inhibit neuroblastoma differentiation by repressing expression of the tumor suppressor in lung cancer–1 (TSLC1) and kinesin family member 1B (KIF1B) in these cells.

In a 2013 study, surface expression of the granulocyte colony-stimulating factor receptor CD114 was found to distinguish a subpopulation of neuroblastoma cells in cell lines and primary tumors, the proportion of which was increased in relapsed tumors. These cells were found to have increased responsiveness to signal transducer and activator of transcription–3 (STAT3), a critical transcription factor controlling neural crest specification. This subpopulation of cells was highly tumorigenic, could self-renew, and could give rise to differentiated progeny with characteristics of premigratory neural crest cells. This subpopulation possessed an undifferentiated phenotype similar to that found in early multipotent neural crest precursors. Further studies are required to understand the role of CSCs in neuroblastoma.

Hereditary Predisposition to Neuroblastoma

A family history has been identified in 1% of cases of neuroblastoma and is suggestive of autosomal-dominant inheritance. In these children, neuroblastoma develops at an earlier age and often manifests as multiple primary tumors.

Genetic Aberrations in Sporadic Neuroblastoma

MYCN Amplification

MYCN, the first oncogene proven to be of clinical significance in neuroblastoma and gene amplification, occurs in approximately 20% to 30% of primary tumors and 90% of cell lines. MYCN amplification is generally associated with advanced disease, poor outcome, and rapid tumor progression in neuroblastoma. It occurs in 5% to 10% of early stage tumors and in half of the tumors of patients with advanced stage disease. The detection of MYCN gene amplification in tumor cells at diagnosis is used by most cooperative groups to stratify patients to more intensive therapy. MYCN copy number appears to be consistent throughout the natural course of the disease, that is, at diagnosis and relapse and between primary and metastatic tumors in individual patients. The Children’s Oncology Group (COG) currently classifies MYCN amplification as more than 10 copies per diploid cell; it is unclear whether lower levels of gain (i.e., MYCN copy number between 3 and 10) confer an adverse outcome. A correlation usually exists between amplification and increased MYCN expression, and tumors with MYCN amplification generally express higher levels of MYCN than do nonamplified tumors; however, it has been reported that high expression is not linked to outcome. MYC deregulation is common in high-risk tumors without MYCN amplification, suggesting that MYC signaling could contribute to the high-risk phenotype.

Amplification manifests cytogenetically as double minutes or homogeneously staining regions; the latter occur mainly in tumors and the former in cell lines ( Fig. 54-2 ). However, there is no difference in clinical outcome whether MYCN amplification is manifested as double minutes or homogeneously staining regions in tumor samples obtained at diagnosis. The genomic region amplified with MYCN is around 500 to 1000 kb, and several genes have been shown to be coamplified with MYCN in neuroblastoma. These genes include DDX1 (DEAD box helicase 1) , NAG ( NBAS [neuroblastoma amplified sequence]), and NCYM (MYCNOS—MYCN opposite strand), and it is possible that coamplification of these genes may contribute to the tumor phenotype.

V-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN) amplification in neuroblastoma.

A fluorescence in situ hybridization image of neuroblastoma tumors depicting MYCN amplification manifested as ( A ) double minutes and ( B ) homogeneously staining regions.

Role of MYCN during Development.

MYCN is a highly conserved bHLH leucine zipper transcription factor located on chromosome 2p24.3 and a member of the myc family of oncogenes. The coding regions of both MYC and MYCN are highly homologous, with each having long 5′ and 3′ untranslated regions and gene products of similar sizes. Both dimerize with Max and bind DNA at consensus E-box sequences (CANNTG; reviewed in reference ). Mice homozygous for disrupted MYCN die at around 10 days of gestation and have multiple organ defects, including tissues of the central and peripheral nervous systems, mesonephros, lung, gut, and heart.

MYCN is highly expressed in fetal and neonatal development, specifically in the forebrain, hindbrain, and kidney, but not in adult tissues. MYCN is expressed in the fetal brain up to the onset of differentiation and is also present at high levels in neuroblasts migrating from the neural crest through the adrenal cortex. MYCN regulates overlapping stem-related gene expression programs in neuroblastoma and neural stem cells. MYCN induces a stemlike state by blocking differentiation pathways and activating self-renewal and pluripotency factors such as KLF2 and KLF4 (Kruppel-like factor 2 and 4) and LIN28B.

Role of MYCN in Tumorigenesis.

Suppression of MYCN expression in neuroblastoma cell lines results in a more differentiated phenotype. Indeed downregulation of MYCN occurs in neuroblastoma cells induced to differentiate with chemical agents, and MYCN overexpression can block retinoic acid–induced differentiation. Deletion of MYCN in neural progenitor cells showed decreased brain size and an increase in neuronal differentiation. Enhanced expression of MYCN and the mutant H- ras gene cause tumorigenic conversion of primary rat embryo fibroblasts, and these transformed cells elicit tumors in athymic mice and isogenic rats. Targeted expression of MYCN to the neuroectoderm of mice using a tyrosine hydroxylase promoter resulted in neuroblastoma tumors with chromosomal copy number abnormalities syntenic to those found in human tumors. The prolonged latency together with the additional chromosomal anomalies in these tumors suggests that additional genetic mutations are required for neuroblastoma formation.

MYCN is involved in all aspects of tumor cell metastasis: adhesion, motility, and invasion (reviewed in reference ). It downregulates integrins α1 and β1 to allow invasion. MYCN-amplified tumors have increased angiogenesis: overexpression of MYCN is associated with proangiogenic factors such as angiogenin and vascular endothelial growth factor (VEGF) through the PI3K/mTOR (mammalian target of rapamycin) pathway.

Role of MYCN in Altered Tumor Metabolism.

Similar to other cancers, neuroblastomas require abnormally high amounts of glucose and other macromolecules to enable their continued unchecked proliferation. These needs are met through alterations in key metabolic pathways that can potentially be harnessed for the development of new anticancer therapies.

Normal cells generate adenosine triphosphate (ATP) from glucose through oxidative phosphorylation when oxygen is abundant. During oxygen deprivation, which occurs commonly in solid tumors, including neuroblastoma, ATP generation occurs through glycolysis (the Warburg effect). Genes involved in glycolysis are selectively activated by hypoxia-inducible factor–1α (HIF1a), which is upregulated in hypoxic states. HIF1a is preferentially expressed in MYCN-amplified neuroblastoma cells; moreover, high levels of MYCN have been shown to override HIF1a inhibitory effects on cell cycle progression, thus enabling continued proliferation under hypoxic conditions. Therefore both deregulated MYCN and HIF1a cooperate to contribute to the Warburg effect in neuroblastoma cells. Moreover, expression of glycolytic genes phosphoglycerate kinase 1 (PGK1) , hexokinase 2 (HK2) , and lactate dehydrogenase A (LDHA) were higher in MYCN-amplified neuroblastoma than in those without MYCN amplification, with depletion of LDHA inhibiting tumorigenesis in vivo, suggesting that these metabolic pathways could be targeted therapeutically.

MYCN-amplified cells have also been shown to depend on exogenous glutamine for their survival. MYCN-amplified neuroblastoma cells overexpress high-affinity glutamine transporters and other glutaminolytic enzymes that correlate with poor prognosis. Glutamine depletion in MYCN-amplified neuroblastoma cells led to apoptosis through p53-independent PUMA (p53-upregulated modulator of apoptosis) and NOXA induction, an effect that is mediated by activation of the activating transcription factor–4 (ATF4). ATF4 has both context-dependent prosurvival and proapoptotic effects. In MYCN-amplified cells, it appears to have a prodeath role, because ATF4 agonists and glutaminolysis inhibitors potently induced apoptosis in vitro and inhibited tumor growth in vivo, suggesting that ATF4 agonists could be potential therapeutics for MYCN-amplified neuroblastomas.

MYCN in Immune Surveillance.

MYCN is also important in immune surveillance in that it represses several chemoattractant proteins that are required for recruitment of natural killer T cells (NKTs). NKTs are important for producing proinflammatory cytokines to recruit immune cells, stimulate the maturation of dendritic cells, and generate antigen-specific T cells to target the tumor. Patients with MYCN-amplified neuroblastoma with bone marrow metastases had fewer bone marrow NKTs. One such protein repressed by MYCN is monocyte che­moattractant protein–1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2). Deletion of MYCN in MYCN-amplified cells rescues MCP-1 production and NKT recruitment. Therefore avenues to improve NKT function are being explored as therapeutic modalities in neuroblastoma.

Chromosomal Abnormalities in Neuroblastoma

Chromosome 11q LOH

Allelic loss of 11q occurs in 35% to 45% of primary tumors. Chromosome transfer experiments involving transfer of an intact chromosome 11 into a neuroblastoma cell line induces differentiation. The common region of deletion has been mapped to 11q23, indicating that this could be a location for a neuroblastoma suppressor gene. Chromosome 11q loss is associated with multiple chromosomal breaks, possibly reflecting an underlying DNA repair defect. Chromosome 11q LOH occurs mainly in tumors without MYCN amplification and identifies a high-risk subset of patients with advanced stage, older age, and disease with unfavorable histologic features. Unbalanced deletion of 11q occurs in 15% to 20% of cases and is associated with a poor event-free survival (EFS) in patients with otherwise low-risk and intermediate-risk disease ( Fig. 54-3 ).

Survival according to unbalanced 11q loss of heterozygosity (unb11q LOH) .

The rates of event-free and overall survival are shown for all patients ( A and B ), for patients whose tumors did not have v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN) amplification ( C and D ), and event-free survival for those with low-risk disease ( E ) and intermediate-risk disease ( F ) as defined by the Children’s Oncology Group (COG) . The numbers of patients at risk for an event are shown along the curves.

(From Attiyeh EF, London WB, Mosse YP, et al: Chromosome 1p and 11q deletions and outcome in neuroblastoma. N Engl J Med 353:2243–2253, 2005.)

Neurotrophin Receptors in Neuroblastoma

Tropomyosin receptor kinase (TRK) receptors, when activated by their respective neurotrophins, mediate the survival and differentiation of neurons during development (reviewed in reference ). NGF-induced dimerization of TRKA (NTRK1) or brain-derived neutrotrophic factor (BDNF)–induced dimerization of TRKB receptors leads to activation of the tyrosine kinase domain, which facilitates binding of SHC and phosphotyrosine binding (PTB) domain protein adaptors and PLCγ protein binding, respectively, leading to activation of the major growth factor–regulated signaling pathways—the Ras/MAPK pathway and PI3K/3-phosphoinositide–dependent protein kinase–1 (PDK1)/AKT pathway—and to increases in Ca++ release and activation of the PLCγ pathway. The main ligand for TRKA, NGF, promotes survival and induces differentiation in developing sympathetic neuroblasts. Neuroblastoma tumor cells with high levels of TRKA expression differentiate in the presence of NGF in vitro but will undergo apoptosis in its absence. Depending on the tumor microenvironment, therefore, TRKA signaling could induce differentiation or regression of favorable neuroblastomas. During normal development, depletion of NGF occurs in sympathetic neurons, causing TRKA signaling to activate predetermined apoptotic pathways. It has been postulated that spontaneous regression of neuroblastomas is but a delay in this normal developmental pattern. A neurodevelopmentally regulated splice variant of TRKA, TrkAIII, has been identified that antagonizes the antioncogenic NGF/TRKA signaling and promotes neuroblastoma tumor growth. High levels of TRKA expression are associated with a good prognosis in neuroblastoma and are strongly correlated with favorable tumor stage, younger age, and nonamplified MYCN . Patients with hyperdiploid tumors with favorable outcome identified by mass screening were also shown to have very high TRKA expression.

The TRKB (NTRK2) transcript is expressed primarily in highly aggressive MYCN -amplified tumors. The ligand for TRKB is BDNF, and activation of TRKB by BDNF leads to enhanced proliferation, migration, angiogenesis and resistance to chemotherapy in neuroblastoma. Neuroblastoma cells that survive repeated exposures to cytotoxic agents express increasing levels of BDNF, suggesting that this pathway contributes to a multidrug-resistant phenotype. Moreover, hypoxia induces increased TrkB expression and may contribute to a phenomenon in the tumor microenvironment where autocrine activation of the BDNF/TrkB signaling enables survival of residual tumor cells after repeated rounds of chemotherapy. The full-length TRKB, which is expressed in about one third of tumors tested, is expressed primarily in those with MYCN amplification, whereas the truncated form resulting from alternative splicing, which lacks the tyrosine kinase domain, is expressed in ganglioneuromas and ganglioneuroblastoma. The truncated TRKB is thought to sequester BDNF and thus prevent TRKB signaling. Studies have shown that survival of neuroblastoma cells exposed to cytotoxic drugs is mediated partly by activation of the Trk receptor via PI3K, activation of Akt, and inactivation of glycogen synthase kinase–3 beta (GSK3B). Thus the response of neuroblastoma cells to chemotherapeutic agents depends on the levels of BDNF; tumor cells expressing low levels of TrkB exhi­bit minimal effects on exposure to cytotoxic agents in the setting of a BDNF-rich environment. BDNF/TrkB-induced increases in HIF1a may lead to increased VEGF and angiogenesis in neuroblastoma. Thus hypoxia-associated increases in neuroblastoma cell invasiveness can be blocked by inhibition of TrkB. Activation of the BDNF/TrkB signaling pathway stimulates tumor cell survival and angiogenesis and contributes to chemotherapy resistance and anoikis.

TRKC, like TRKA, is involved in the biology of favorable neuroblastomas, and expression corresponds to lower stages. Although its expression is found in 25% of primary neuroblastomas, it does not have independent prognostic significance, because all tumors with TRKC expression also have TRKA expression.

Apoptosis

In contrast to most human tumor types, the p53 gene is very rarely mutated in neuroblastoma at diagnosis but is found in chemotherapy resistance. Other mechanisms of p53 loss of function, such as cytoplasmic sequestration, mouse double minute 2 (MDM2 protooncogene) amplification, or TWIST-mediated suppression have been proposed, but their contribution appears to be limited. Although basal p53 expression in neuroblastoma cells is largely confined to the cytosol, p53 protein levels were found to increase mainly in the nucleus after radiation-induced DNA damage.

Epigenetic modification of the pro-apoptotic gene caspase-8 has been observed in neuroblastomas. Up to 70% of human neuroblastoma cell lines and 25% of primary tumors tested lack caspase-8 expression and fail to undergo apoptosis. Loss of expression of both caspase 8 (apoptosis-related cysteine peptidase, CASP8 ) alleles is correlated with methylation of the caspase-8 gene, and treatment with the demethylating agent decitabine restored caspase-8 expression and increased susceptibility to doxorubicin-induced apoptosis. In addition, methylation has been shown to be the mode of silencing of other genes involved in apoptosis: the four tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) apoptosis receptors and the caspase-8 inhibitor FLICE-like inhibitory protein (FLIP).

Overexpression of BCL2, an inhibitor of apoptosis, has been found to be associated with unfavorable histology and MYCN amplification. Neuroblastoma cells and tumors are primed for death with sequestration of BIM, a direct activator of apoptosis, either by the prosurvival BCL-2 or myeloid cell leukemia–1 (MCL-1) proteins. This pattern of survival dependency has been shown to predict the pattern of response to Bcl-2 antagonists. The Bcl-2/Bcl-xl/Bcl-w inhibitor ABT-737 was active against Bim : Bcl-2 primed tumor xenografts but not against cells where Bim was sequestered by Mcl-1. In isogenic cell lines at diagnosis and relapse, therapy resistance was shown not to be mediated by upregulation of Bcl-2 family members or loss of Bim priming but by repression of Bak and/or Bax-mediated apoptosis.

Therapy Resistance

The multidrug resistance gene encoding P-glycoprotein is thought to function by causing enhanced drug efflux from the cell, and its overexpression has been found to predict outcome of therapy for neuroblastoma. The multidrug resistance-associated protein (MRP), like P-glycoprotein, mediates resistance to a number of drugs. MRP is expressed by neuroblastoma tumors of all stages. Tumors with MYCN amplification have been shown to have significantly higher expression levels of MRP than do those with normal MYCN copy numbers. Reduced MRP expression levels correlate with differentiation of neuroblastoma cells in vitro. In addition, an association has been found between high levels of MRP expression and poor outcome, which is independent of MYCN amplification. It is possible that both multidrug resistance protein–1 (MDR1) and MRP function together with several other factors that confer resistance to drug therapy in neuroblastoma such as MYCN amplification, TRKB signaling, or loss of p53 expression.

MYCN-amplified tumors were found to possess a population of cells with CSC characteristics that was enriched during prolonged drug selection. Treatment with the histone deacetylase (HDAC) inhibitor vorinostat led to histone acetylation and increased the sensitivity of these cells to chemotherapy, with loss of their stemness properties and downregulation of stemlike marker genes.

Tumor Angiogenesis

Neuroblastoma is characterized by prominent angiogenesis, and neuroblastoma cells have been shown to induce angiogenesis in the chick embryo chorioallantoic membrane assay. Increased tumor vascularity and microvascular proliferation is correlated with widely disseminated disease, MYCN amplification, unfavorable histology, and a poor outcome. Such aggressive tumors are associated with high expression of VEGF, basic fibroblastic growth factor, and platelet-derived growth factor A and integrins, which are markers of active angiogenesis. The presence of angiogenesis appears to be influenced by the cellular composition of the tumor in that in highly aggressive stroma-poor neuroblastoma, angiogenesis is present as a result of the secretion of angiogenic stimulators, whereas in stroma-rich tumors, numerous angiogenic inhibitors secreted by Schwann cells appear to maintain the inhibitory phenotype. The Schwann cells in neuroblastoma tumors have been shown to have very low tumor vascularity with production of angio­genesis inhibitors, such as tissue inhibitor of matrix metalloproteinase–2 (TIMP-2), pigment epithelium-derived factor, and SPARC (secreted protein acidic and rich in cysteine), a calcium-binding matricellular glycoprotein. SPARC expression has been found to be inversely correlated with the degree of malignant progression in neuroblastoma tumors, and neutralizing SPARC with antibodies reverses the antiangiogenic activity of Schwann cell–conditioned media.

Metastasis

Metalloproteinases such as matrix metalloproteinase–9 (MMP9), along with CD44 and NM23-H1, which regulate tumor cell adhesion and migration, may play a role in metastasis. Overexpression of MMP2 and MMP9 is associated with tumor invasion and metastasis in many types of cancer, whereas inhibitors of MMPs have been shown to suppress tumor invasion and angiogenesis. An association between increased levels of MMP2 and MMP9 and advanced-stage tumors has been observed in neuroblastoma. Caspase-8 has also been shown to be a metastasis suppressor gene. Decreased caspase-8 expression has been shown to occur during the establishment of neuroblastoma metastases in vivo, and reconstitution of caspase-8 expression in deficient neuroblastoma cells suppressed these metastases. Caspase-8 selectively potentiated apoptosis in metastasizing cells, and loss of caspase-8 allowed cellular survival in the stromal microenvironment and promoted metastases. In the TH-MYCN mouse model of neuroblastoma, caspase-8 was associated with increased metastasis, specifically to the bone marrow, possibly caused by upregulation of genes involved in epithelial-mesenchymal transition (EMT), decreased cell adhesion, and increased fibrosis.

Localized Disease

Approximately 40% of patients present with localized disease. Primary abdominal disease can present as an asymptomatic abdominal mass or with abdominal pain and fullness. Symptoms of obstruction can occasionally be seen, along with renin-mediated hypertension caused by compression of the renal vasculature. Abdominal or pelvic tumors can also occasionally cause compression of lower extremity venous and lymphatic drainage, leading to lower extremity and scrotal swelling. Rarely abdominal tumors spontaneously hemorrhage, and patients with such hemorrhage present with a sudden, dramatic enlargement of an abdominal mass with increased dis­tension and pain. Lower thoracic tumors are usually identified incidentally when a chest radiograph is obtained for unrelated reasons. Occasionally large thoracic tumors are associated with mechanical obstruction and resultant superior vena cava syndrome. Upper thoracic or cervical tumors may cause Horner syndrome. Congenital tumors arising in this site can also cause hematochromia of the iris as a result of decreased pigmentation of the iris on the affected side. Neuroblastoma arising from the paraspinal ganglia in the chest, abdomen, or pelvis can grow through the intervertebral foramina and compress the spinal cord. Patients may be asymptomatic or present with pain or neurologic deficits resulting from spinal cord compression, which requires emergent treatment as detailed later in this chapter.

Metastatic Disease

Metastatic spread of neuroblastoma occurs through the lymphatics or hematogenously. Regional lymph node metastases are seen in one third of patients with apparently localized tumors, and half of patients present with hematogenous metastases. Hematogenous spread occurs most frequently to the bone, bone marrow, and/or liver; rarely, metastasis to the lungs and brain occurs, usually at relapse rather than at presentation. Classic signs of metastatic neuroblastoma include proptosis and periorbital ecchymoses (commonly referred to as “raccoon eyes”) due to tumor infiltration of the periorbital bones. Patients also frequently present with limping and irritability due to bone pain from bone and bone marrow disease, as well as nonspecific symptoms including fever and failure to thrive. The presence of fever is usually associated with extensive bone metastases. Signs and symptoms of bone marrow replacement are also sometimes seen—most frequently pallor (which may also be caused by bleeding within the primary tumor), as well as bruising and increased risk of infection as a result of a low white blood cell count.

Stage 4S Neuroblastoma

Stage 4S (S = special) is a unique presentation of neuroblastoma seen in infants; it was originally described by D’Angio, Evans, and Koop in 1971. Infants with stage 4S neuroblastoma often present with massive hepatomegaly caused by diffuse liver metastases and nontender, bluish subcutaneous nodules as a result of skin nodules. Stage 4S disease accounts for 7% to 12% of neuroblastoma diagnoses and is historically defined as the presence of a small, localized primary tumor with metastases isolated to the liver, skin, and/or bone marrow in an infant younger than 12 months. This special neuroblastoma often spontaneously regresses, although infants younger than 2 months can present with respiratory compromise due to a rapidly enlarging liver and may require cancer-directed treatment. Based on an analysis of a large cohort of international patients diagnosed with neuroblastoma between 1990 and 2002, the INRG Task Force has extended the definition of 4S neuroblastoma to include patients up to the age of 18 months and those with primary tumors crossing the midline.

Paraneoplastic Syndromes

Opsoclonus-myoclonus syndrome (OMS) and vasoactive intestinal peptide syndrome, which are well-described paraneoplastic syndromes associated with neuroblastoma, are detailed in this section, along with a newly described syndrome known as rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD). Patients with these syndromes usually have localized tumors with favorable biology, perhaps because the syndrome causes early detection of tumor or, more likely, because of a correlation between the syndrome and the tumor biology.

OMS is observed in 2% to 3% of children with neuroblastoma and is usually diagnosed between the age of 1 and 3 years. It is often referred to as “dancing eyes, dancing feet syndrome” and is characterized by the acute onset of rapid eye movements, ataxia, myoclonic jerking of the limbs and trunk, and behavioral disturbances. Although children with neuroblastoma and OMS generally have favorable tumor prognostic features and a high survival rate, 70% to 80% experience long-term neurologic sequelae that can include global developmental delay, speech and/or motor delay, behavioral disturbances, and cognitive deficits that can seriously affect quality of life. OMS in children is associated with neuroblastoma more than half of the time. After exclusion of CNS pathology, all children with OMS should be evaluated for neuroblastoma with imaging of their chest and abdomen, an iodine 123–metaiodobenzylguanadine (MIBG) scan, and urine catecholamines. Traditional methods to detect neuroblastoma may be insensitive in patients with OMS, and therefore high-resolution computed tomography (CT) or magnetic resonance imaging (MRI) of the chest and abdomen should be considered, even in patients with negative catecholamines or negative results of an MIBG scan, to screen for small tumors.

OMS and its sequelae are thought to be immune mediated and are likely caused by the presence of an antineural antibody that cross-reacts with a common antigen on both neuroblastoma and normal nervous system tissue. Improvement in symptoms can be seen after tumor removal in some cases, but generally immunosuppression with glucocorticoids or adrenocorticotropic hormone is used to relieve acute symptoms. However more than 80% of patients will have a relapse of symptoms with steroid weaning or in association with a viral syndrome, and more than half of patients will need prolonged steroid treatment, necessitating additional treatment approaches. Intravenous immune globulin has also been used in the treatment of OMS, either with or without steroids, with varying success. Case reports have shown efficacy of other immunomodulating strategies including plasmapheresis, rituximab, and mycophenolate. Although often effective in treating acute symptoms, none of these treatments has been consistently correlated with improved long-term outcome. Interestingly, a report from the Pediatric Oncology Group (POG) suggested that patients who received chemotherapy to treat their neuroblastoma had a more favorable neurologic outcome. This benefit may be due to the immunosuppressive effects of chemotherapy or to less severe activation of the immune system in patients with more advanced stages of tumor. The same benefit was not observed in a similar review performed by the Children’s Cancer Group (CCG), and the benefit of chemotherapy for the treatment of OMS needs to be investigated further. The COG recently conducted a trial for OMS in which patients with low-risk neuroblastoma who would not otherwise receive chemotherapy were treated with cyclophosphamide in addition to steroids. Long-term outcome of these patients compared with historic control subjects should provide insight regarding the role of chemotherapy for the treatment of OMS.

Further investigation is needed to determine the best treatment approach for OMS. Regardless of treatment, physicians caring for patients with OMS should anticipate long-term neurologic abnormalities and use early intervention to help minimize these deficits.

Tumor secretion of vasoactive intestinal peptide can cause a syndrome of chronic watery diarrhea and failure to thrive. Most tumors secreting vasoactive intestinal peptide are histologically mature. Surgical removal of the tumor usually results in complete resolution of symptoms.

ROHHAD syndrome is characterized by rapid-onset obesity, hypothalamic dysfunction, hypoventilation, and autonomic dysregulation. Tumors of neural crest origin have been found in more than one third of cases and are usually differentiated (ganglioneuromas or ganglioneuroblastomas). ROHHAD syndrome most commonly affects otherwise normal children between 2 and 4 years of age and typically presents first with rapid weight gain and obesity followed at varying time intervals by autonomic dysfunction and hypoventilation. The prognosis is poor, and at least 25% of children die because of respiratory failure. A genetic cause has been suggested because of the constellation and consistency of symptoms and the reported occurrence in siblings, but several candidate genes (including PHOX2B, ASCL1 [achaete-scute family bHLH transcription factor–1] , NTRK2 [TRKB] , and BDNF ) have been sequenced in affected patients and have not been informative. Monozygotic twins discordant for ROHHAD have recently been reported, although this in itself should not preclude a genetic cause because modifier genes or epigenetic variation may play a role in the variable phenotype. Because of the association with neuroblastic tumors and the finding of extensive infiltrates of lymphocytes and histiocytes in the hypothalamus of some patients, a paraneoplastic cause has been postulated. Immune-modulating agents including intravenous immunoglobulin, rituximab, and cyclophosphamide have been tried in small series of patients with varying success, and collaborative studies are needed to determine the cause of this rare disorder. Surveillance for tumors in patients with this constellation of features is recommended.

Diagnosis

The diagnosis of neuroblastoma is usually established from histopathologic evaluation of primary tumor tissue. In most cases, especially if features of neuronal differentiation are present, a tissue diagnosis of neuroblastoma can be made on the basis of conventional hematoxylin and eosin staining. However, in cases in which there is little differentiation and only small round blue cells are evident, immunohistochemical staining for neuron-specific enolase, chromogranin A, and/or synaptophysin, as well as cytogenetic and molecular analysis, can help differentiate neuroblastoma from other small round blue cell tumors of childhood.

In addition to establishing the diagnosis, primary tumor material is essential for risk stratification and prognosis, particularly in children younger than 18 months with locoregional or metastatic spread. In cases in which primary tumor tissue cannot be obtained safely, the diagnosis of neuroblastoma can also be made by demonstrating unequivocal neuroblastoma cells in a bone marrow aspirate or biopsy in conjunction with increased urinary catecholamines. Urinary catecholamine metabolites are increased in 90% to 95% of neuroblastomas using sensitive detection techniques such as high-performance liquid chromatography and can be helpful in establishing a diagnosis, as well as in monitoring disease activity and response to therapy in patients who are known to excrete the metabolites. In sympathetic cells the catecholamine precursor 3,4-dihydroxyphenylalanine (DOPA) is converted to dopamine by DOPA decarboxylase, which is converted to norepinephrine and then to epinephrine by the enzyme phenylethanolamine-N-methyltransferase, which is not present in neuroblastoma cells. Instead the enzymes catechol-O-methyltransferase and monoamine oxidase convert DOPA and dopamine to homovanillic acid and norepinephrine and epinephrine to vanillylmandelic acid, both of which are inactive metabolites that are excreted in the urine. Both urinary homovanillic acid and vanillylmandelic acid should be measured for diagnostic purposes. For undifferentiated tumors, urinary dopamine may also be useful. Despite this catecholamine production, symptoms of catecholamine excess such as hypertension, flushing, and sweating are rarely seen in persons with neuroblastoma, likely because of the relatively low concentrations of active catecholamines in the circulation.

Clinical Disease Assessment

The primary tumor and the extent of disease should be evaluated using imaging techniques both at diagnosis and to evaluate response to treatment. Either CT or MRI are routinely used to evaluate the extent and origin of the primary tumor, as well as possible solid organ metastases. CT is more widely available and can rapidly capture images, reducing the need for sedation, but it is associated with substantial radiation exposure. Ionizing radiation is not used to perform MRI but it has a longer imaging acquisition time, making sedation necessary in most young children. Currently no consensus exists with regard to which imaging technique is optimal for evaluating tumors and metastases in the abdomen, pelvis, or mediastinum. Availability and clinical factors, including the risks of sedation, should be taken into account. MRI is preferred for assessing paraspinal lesions, particularly those with possible intraextension and spinal cord impingement. Although abdominal ultrasonography is not as useful for assessing tumor volume and anatomy, it may be useful as a noninvasive method for following tumor response or surveying for disease relapse. Brain imaging is only recommended if clinically indicated by symptoms or examination.

Bone and bone marrow should also be evaluated by imaging to look for the presence and extent of metastatic disease. MIBG is a norepinephrine analogue that is selectively concentrated in more than 90% to 95% of neuroblastomas and can be combined with either the iodine 131 or I isotopes for scanning purposes. MIBG scintigraphy is highly sensitive and specific for detecting osteomedullary disease and can also be used to assess the primary tumor, as well as occult soft tissue disease ( Fig. 54-5 ). The I isotope provides enhanced image resolution and is the isotope of choice when it is available. MIBG scans are essential for the initial staging of neuroblastoma and should be performed prior to primary tumor resection. International guidelines are available for patient preparation, radiotracer administration, and imaging techniques. A bone scan using technetium 99m–diphosphonate scintigraphy is a relatively sensitive but less specific method to detect bony metastases in patients with neuroblastoma. Bone scans are recommended in cases in which the primary tumor is not MIBG avid, or in cases in which tumor uptake of MIBG cannot be confirmed (i.e., in cases in which the primary tumor is removed prior to imaging for metastatic disease). Fluorine 18–labeled deoxyglucose positron emission tomography (FDG-PET) can also be used to evaluate metastatic disease in persons with neuroblastoma. Its sensitivity and specificity in comparison with MIBG scintigraphy is still being evaluated, with studies suggesting that MIBG is more sensitive than FDG-PET for the detection of bone lesions and skull-based lesions, and that FDG-PET is more sensitive than MIBG for the detection of small soft tissue tumors and nodal metastases. The routine use of FDG-PET is not currently recommended, but it may be a helpful tool to follow-up patients with metastatic disease who do not have MIBG-avid tumors.

Metaiodobenzylguanidine scan.

Primary tumor uptake (right adrenal gland), as well as bone/bone marrow metastases, are visualized.

(Courtesy of F. Grant, MD, Division of Nuclear Medicine, Children’s Hospital, Boston, Mass.)

Bone marrow aspirates and biopsies from both iliac crests are recommended to assess for the presence of bone marrow disease using standard histologic analysis. Both immunocytochemical and polymerase chain reaction–based technologies can be used to increase sensitivity of marrow detection. Immunocytochemical analysis of bone marrow aspirates with monoclonal antibodies directed against neural-specific antigens (e.g., GD2 and NCAM, neural cell adhesion molecule) increases the sensitivity of detecting marrow involvement to 1 : 100,000 nucleated cells. Reverse-transcriptase polymerase chain reaction methodologies that target the expression of neuroblastoma-specific messages such as tyrosine hydroxylase, protein gene product 9.5, or GD2 synthase can enhance sensitivity further. However, the clinical and prognostic significance of this enhanced detection, remains to be determined, and these studies are generally only recommended within the context of a clinical study.

Staging

The International Neuroblastoma Staging System (INSS) was established in the 1990s to replace three major staging systems used throughout the world in an effort to provide international uniformity so that clinical trials and biology studies performed by different groups in different countries could be compared. The INSS ( Table 54-3 ) is largely based on the surgical assessments of a patient at diagnosis and has been used nationally and internationally for more than 20 years. However, because surgical approaches are not uniform, the INSS stage for patients with locoregional disease can vary considerably depending on the degree of initial tumor resection, making comparison of clinical trials conducted in different parts of the world difficult. To address this problem, the INRG Task Force developed a new staging system based on tumor imaging rather than extent of surgical resection. This staging system (known as the INRG staging system) uses radiologic characteristics of the primary tumor to predict surgical risk and resectability ( Tables 54-4 and 54-5 ). Using this approach, in INRG staging, locoregional disease is classified as stage L1 or L2 on the basis of whether the tumor is locally invasive by imaging at the time of diagnosis. Stage M indicates metastatic disease that is assessed in the same way it was assessed by INSS criteria, via both imaging and pathology.

The INRG system also differs from the INSS system in that the midline and lymph node status are not included in the staging criteria. In addition, INRG stage MS includes patients up to 18 months of age and those with L2 tumors, whereas INSS stage 4S has an upper age limit of 12 months and includes only patients with stage 1 or 2 primary tumors.

Because imaging can be retrospectively reviewed, the INRG staging system should provide data that are more uniform than the INSS system. The INRG staging system is currently used in Europe and will be adopted into the COG studies as well.