Genetic Predisposition

The incidence of familial NB is estimated at 1% to 2%. Cases often involve multifocal and/or bilateral adrenal primary tumors with a median age of onset of 9 months. The pattern of inheritance is autosomal dominant with incomplete penetrance. NB can occur in patients with other neural crest disorders, such as Hirschsprung disease (HSCR), congenital central hypoventilation syndrome (CCHS), and neurofibromatosis type 1 (NF1). Mutations in the Phox2b homeobox gene have been detected in subsets of patients with familial NB and usually are associated with other neurocristopathies, such as HSCR and CCHS. Phox2b mutations have also been detected in approximately 2% of sporadic NB. There are many reports of NB in patients with NF; however, there are conflicting data as to whether germline NF1 mutations are associated with an increased risk to develop NB.

Linkage studies in familial NB pedigrees identified candidate chromosomal predisposition regions including 16p, 12p, and 2p and led to the identification of germline mutations in the tyrosine kinase domain of the anaplastic lymphoma kinase ( ALK ) oncogene. ALK is involved in nervous system development, and central nervous system (CNS) anomalies have been reported in some patients with germline ALK mutations. Sporadic NB tumors also harbor ALK abnormalities, including genomic amplification (2%–3%) and missense mutations (8%–12%) (see “Somatic Gene Mutations”), that can be targeted by pharmacologic inhibitors. Studies of ALK inhibitors in NB and other tumors with ALK aberrations (eg, anaplastic large cell lymphoma) have shown promising results. NB cases are also detected in other familial cancer syndromes, including Beckwith-Wiedemann syndrome, Li-Fraumeni, Noonan ( PTPN11 ), some subtypes of Fanconi anemia, and some chromosomal breakage syndromes.

Recent genome-wide association studies using peripheral blood from thousands of patients with NB have also identified germline genetic variants that may predispose to the development of sporadic NB. These variants include single nucleotide polymorphisms (SNPs) in LINC00340, BARD1, LMO1, DUSP12, DDX4, LIN28B , HACE1 , and TP53 . Unlike the rare germline mutations in ALK and Phox2B described earlier, these SNPs are more frequent but individually have less dramatic impacts on the NB risk. The interplay between multiple germline variants and somatic alterations, discussed later, may influence the initiation and progression of NB.

Symptoms

NB presentations vary based on the disease extent and tumor location, which may occur anywhere along the sympathetic chain resulting in local effects on organs, vessels, or nerves ( Fig. 2 , Table 1 ). Most of them (65%) arise in the abdomen, most commonly the adrenal gland, and may be asymptomatic or associated with hypertension, abdominal pain, distension, and constipation. Other sites include the neck, chest, and pelvis. The primary site location is associated with age and outcome. Cervical and thoracic tumors are more common in infants and may present with Horner syndrome (unilateral ptosis, anhidrosis, and myosis) and respiratory symptoms. Epidural or intradural tumor extension occurs in 5% to 15% of patients and may result in spinal cord compression and paraplegia. Two rare paraneoplastic syndromes associated with NB include secretory diarrhea caused by tumor production of vasoactive intestinal peptide and opsoclonus myoclonus ataxia syndrome (OMS). OMS is reported in 2% to 3% of patients and is commonly associated with favorable well-differentiated tumors. OMS is characterized by myoclonic jerks and random eye movements with or without ataxia, is attributed to immune-mediated effects, and often persists after resection, resulting in significant neurodevelopmental sequelae.

Percent distribution of NBs by primary site and age; Surveillance, Epidemiology and End Results Program (1975 to 1995).

( Adapted from Ries L, Smith M, Gurney J, et al. Cancer incidence and survival among children and adolescents: United States SEER Program 1975–1995, National Cancer Institute, SEER Program. Bethesda (MD): NIH Pub; 1999. p. 99–4649. NIH Publication No. 99–4649.)

Approximately half of patients present with localized or regional disease, and 35% have regional lymph node spread at the time of diagnosis. Distant metastases are detected in 50% of patients at diagnosis and occur through both lymphatic and hematogenous routes. The most common sites include bone, bone marrow, and liver. NB has a particular predilection to spread to metaphyseal, skull, and orbital bone sites, resulting in a classic presentation characterized by periorbital ecchymoses (raccoon eyes), proptosis, and potentially visual impairment. In contrast to the frequent lack of symptoms for locoregional tumors, patients with widespread disease are often ill appearing with fever, pain, irritability, and weight loss. Less common sites of metastases at diagnosis include the lung and brain; however, CNS disease at relapse is increasingly common. In infants there is an unusual pattern of metastases, stage 4S or MS (see “Staging” later), characterized by skin nodules and/or diffuse liver involvement and hepatomegaly often associated with respiratory compromise.

Diagnosis is confirmed either by (1) tumor tissue biopsy and histopathology ( Fig. 3 ) or (2) a combination of NB tumor cells detected in bone marrow together with elevated urine or serum catecholamine or catecholamine metabolites (dopamine, vanillylmandelic acid, and homovanillic acid). Evaluation includes cross-sectional imaging with computed tomography or MRI to determine size, regional extent (including intraspinal invasion), distant spread to neck, thorax, abdomen and pelvis (see Fig. 4 ). Bilateral iliac crest bone marrow aspirates and biopsies are required to determine tumor involvement by histology. Radioiodine-labeled metaiodobenzylguanidine (MIBG), a norepinephrine analogue that selectively concentrates in sympathetic nervous tissue, is used to detect primary tumors and metastatic sites. Approximately 90% of patients have MIBG-avid disease, and semiquantitative scoring systems are being integrated into NB response criteria. [(18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) scans are recommended for detecting metastatic disease in patients whose tumors are not MIBG avid. Technetium bone scans can be used to detect cortical bone disease if MIBG and PET scan are not available.

Histopathology and fluorescence in situ hybridization (FISH) assays ( A – C ). Shown are representative images (hematoxylin-eosin, original magnification ×200 [ A and C ] ×400) from 3 different histologic appearances of NB: ( A ) poorly differentiated NB, ( B ) differentiating NB, and ( C ) ganglioneuroblastoma (stroma-rich NB). The fluffy pink material separating the cells is neuropil (categorized as stroma-poor). ( A ) The poorly differentiated NB cells have minimal cytoplasm, discernible only as purple-stained nuclei. ( B ) The neuroblasts are differentiating as reflected by defined pink cytoplasm and larger nuclei. ( C ) The neuroblasts have the features of fully differentiated ganglion cells, and the spindle cell areas in the 4 corners are composed of Schwann cells (categorized as stroma-rich). ( D ) FISH showing MYCN amplification (MYCNA). The presence of multiple copies of MYCN is detected in tumor cells using a labeled probe ( red ) for the chromosomal location 2p region that includes the MYCN gene. MYCNA is defined as greater than 10 copies. ( E ) FISH showing 1p loss of heterozygosity (LOH). Cells show 2 signals from the control 1q probe ( green ) and 1 signal for the 1p 36 probe ( red ) indicating that there is loss of one copy of 1p36 loci (LOH) and 2 normal copies of 1q.

( Courtesy of Dr Paul Thorner, Pathology Department, and Dr Mary Shago, Cytogenetics Laboratory, Hospital for Sick Children, Toronto.)

Diagnostic imaging of NB. Shown are representative images of NB tumors from different primary locations from diagnostic evaluations. ( A ) Computed tomography (CT) scan (axial view) shows a typical retroperitoneal mass arising from the adrenal with calcifications (white speckles in tumor mass, black arrows ) and tumor encasement of vessels (aorta, white arrow ). The left kidney demonstrates mild pelviectasis, which is commonly seen secondary to the mass effect. ( B ) CT scan (coronal view) of very large liver with multiple NB tumor nodules (darker than surrounding liver parenchyma), which is typically seen in infants with International Neuroblastoma Staging System stage 4S/INRG MS. ( C ) MRI scan (sagittal view) shows a paraspinal thoracic mass ( arrows ) with intraspinal extension and spinal cord compression. ( D ) Brain and orbital CT (axial) with large metastases involving the orbits, with more extensive involvement on the left ( arrow ). ( E ) I-123 metaiodobenzylguanidine scan demonstrates widespread bony metastases in the extremities, vertebrae, and pelvis (darker lesions). Note the normal physiologic uptake in the heart, liver, and bladder.

Staging

Until recently, the criteria for diagnosis and staging were based on the surgical-pathologic International Neuroblastoma Staging System (INSS) ( Box 1 ). INSS stages 1 to 3 are localized tumors that are classified based on the amount of resection, local invasion, and node involvement. Stage 4 is defined as distant metastases; 4S (4Special) is characterized by metastases to the liver, skin, and/or marrow in infants, which is usually associated with favorable biological features and can undergo spontaneous regression. In 2009, the International Neuroblastoma Risk Group’s (INRG) stratification system was developed by representatives from a major consortium in North America (Children’s Oncology Group [COG]) Europe (SIOPEN, International Society of Pediatric Oncology European Neuroblastoma), and Germany, Japan, and Australia. The INRG staging system (INRGSS) uses surgical risk factors (SRFs), which are preoperative radiological features to distinguish locoregional tumors that do not involve local structures (INRGS L1) from locally invasive tumors with imaging-defined risk factors (IDRFs) (INRGS L2) ( Boxes 2 and 3 ). INRGS M and MS refer to tumors with distant metastases and have the INSS 4 or 4S pattern of spread, respectively.

Stage and Age

Many studies have consistently demonstrated the independent prognostic value of the INSS stage, including an INRG database analysis that reported superior event-free survival (EFS) and OS for patients with nonmetastatic NB (INSS stages 1, 2, and 3) and INSS stage 4S (83 ± 1% and 91 ± 1%) compared with only 35 ± 1 and 42 ± 1%, for patients with INSS stage 4 disease. With the exception of stage 4S, specific metastatic sites have not been incorporated into staging systems. However, retrospective studies suggest that spread confined to distant lymph nodes may predict improved outcomes, whereas there is a trend toward inferior outcomes for patients with metastases to the lung or bone marrow. Although retrospective studies have demonstrated the prognostic significance of the INRGSS, which incorporates SRFs, this will be prospectively validated across North America and Europe.

Age was one of the first prognostic indicators identified. In comparison to infants, patients older than 1 to 2 years at diagnosis have an inferior outcome; this effect is more prominent for patients with metastatic disease. Historically, a cutoff of 365 days had been used as a surrogate for tumor behavior; however, London and colleagues studied the continuous nature of age for 3666 patients and concluded that the most prognostic cutoff was 460 days (15.1 months). Several retrospective studies specifically examined whether 18 months might represent a more clinically relevant cutoff and demonstrated that EFS and OS for patients with INSS stage 4 disease aged 12 to 18 months (with favorable tumor biology) was similar to that of patients aged less than 12 months. Similarly, patients with INSS stage 3 disease aged 12 to 18 months had a superior outcome to those older than 18 months. Prospective COG trials will determine whether reduction of therapy for toddlers aged 12 to 18 months with biologically favorable tumors, traditionally treated with more intensive regimens, will still provide superior outcomes. Older children, adolescents, and young adults with NB have a more indolent course and worse overall outcome despite infrequent MYCN oncogene amplification ( MYCNA ); however, no specific prognostic age cutoffs greater than 18 months have been identified.

Histopathology

Pathologic characteristics have been used to further classify tumors into favorable and unfavorable categories, initially using a system developed by Shimada and colleagues that provided the basis for the more recently revised International Neuroblastoma Pathology Committee (INPC) criteria. The prognostic value of INPC classification, based on age, presence of Schwannian stroma, grade of neuroblastic differentiation, and Mitosis-karyorrhexis index, has been validated in large cooperative group studies to identify specific patient risk groups that may benefit from modified therapy. In the COG P9641, patients with INSS stage 1 and 2 disease with favorable histology had a significantly better outcome than those with unfavorable histology (UH) (EFS 90 ± 3% and 72 ± 7%, OS 99 ± 1% and 86 ± 5%).

Tumor Genetics

NB genetic features have been used for risk stratification for more than 20 years. Two broad categories of genetic aberration patterns include (1) tumors with whole chromosome gains, lack of structural changes, and hyperdiploid karyotype and (2) tumors with segmental chromosomal aberrations (SCAs) and diploid DNA content, which are often associated with poor outcomes. SCAs often include partial gains and losses of chromosomal regions predicted to encode oncogenes and tumor suppressors, respectively.

Allelic Gains, Amplifications, and Oncogenes

MYCNA defined as greater than 10 copies is detected in approximately 20% of NB tumors, with a higher incidence in INSS stages 3 and 4 (40%) but only 5% of stages 1, 2, and 4s. Many studies have demonstrated that in comparison to patients with non- MYCNA tumors, patients with MYCNA have a significantly worse outcome (reviewed in Refs. ). All patients with MYCNA stage 3, 4 and 4S tumors are classified as HR, including infants; however, the prognostic significance of MYCNA in rare cases of localized resected NB remains controversial. Importantly, most laboratory animal models for NB rely on overexpression of MYCN in neural crest cells ; recent studies have identified drugs that target MYCN to inhibit NB growth.

Amplification of ALK , located at 2p23 in close proximity to MYCN at 2p24, is detected in 2% to 3% of NB and is more common, though not exclusively, observed in tumors with MYCN amplification. A 17q gain detected in greater than 60% of tumors is often associated with other poor prognostic markers (eg, MYCNA , older age). Although the specific genes that may have oncogenic roles on 17q have not been identified, candidates include BIRC5 (survivin), PPMID (WIP1), and NME1/2 .

Allelic Losses and Tumor Suppressor Genes

The most frequently deleted chromosomal regions in NB include 1p, 4p, 11q, and 14q. Chromosome 1p loss of heterozygosity (LOH), detected in 23%–30% of tumors, predicts poor outcome. 1p36 LOH correlates with MYCNA and other HR features (eg, metastasis, age >1, UH), and thus, 1pLOH may be most relevant as an independent prognostic factor in infants and patients without MYCNA . The 1p candidate tumor suppressor genes include the chromatin remodeling protein CHD5 and transcription factor CASZ1. Chromosome 11qLOH is detected in approximately 30% to 40% of patients. Like 17q gain and 1pLOH, 11qLOH is more common in patients with stage 4 disease and predicts poor prognosis; however, 11qLOH is rarely associated with MYCNA and, therefore, may predict additional HR subsets within the non- MYCNA tumors. One 11q candidate gene, CADM1 , has been implicated in NB growth and proliferation. Although INRG currently includes 11qLOH as criteria to upstate to HR classification, prospective trials are ongoing to determine whether 11qLOH predicts poor outcomes for non-HR patients.

Segmental Chromosome Aberrations

Historically, individual chromosomal loci were analyzed using polymerase chain reaction or fluorescent in situ hybridization–based assays. Recent studies using techniques that assess the whole genome, such as comparative genome hybridization and SNP arrays, demonstrate that it is the genomic pattern and not individual losses/gains that is most prognostic. Tumors with numerical chromosomal abnormalities (NCAs) characterized by whole chromosome gains and losses have an excellent outcome, even in patients greater than 18 mo. In contrast, patients with segmental chromosome aberrations (SCA), characterized by gains and losses of smaller fragments, have an inferior outcome. SCAs may be a particularly strong predictor of poor outcome in infants with locally unresectable or metastatic non- MYCN amplified tumors. Prospective trials in North America and Europe will determine whether the presence of SCAs (≥1 of the following: segmental loss at 1p, 3p, 4p, 11q or gain at 1q, 2p, or 17q) can distinguish less favorable subsets of patients within the non-HR groups of patients and potentially replace tests that detect single gene losses/gains.

DNA Content

Ploidy or tumor DNA content (chromosome number) is a powerful predictor of survival. Hyperdiploid tumors (DNA index >1) with an increased amount of DNA in comparison with diploid tumors (DNA index = 1) are associated with a more favorable prognosis. Ploidy is most prognostic in infants and patients with localized disease and has been used prospectively to inform risk assignment and tailor therapy for patients with non-HR NB.

Somatic Gene Mutations

Recently, next-generation sequencing approaches have revealed that, in contrast to adult carcinomas, there is a striking lack of recurrent NB tumor (somatic) mutations. The most commonly mutated gene is ALK (8%–10%), with an additional 3% harboring ALK amplification. ALK genomic aberrations are detected in all risk groups and are associated with an adverse outcome, and high levels of ALK protein or amplification may correlate with poor outcomes independent of mutation status. Mutations in ATRX (alpha thalassemia/mental retardation syndrome X linked), which is involved in telomere maintenance, are detected more frequently in older patients with NB. Deletions and point mutations of the chromatin remodeling proteins AT-rich interactive domain 1A and B ( ARID1a/1b ) were detected in 11% of tumors. Other mutations detected in less than 5% of tumors include MYCN, TP53 , PTPN11 , and genes involved in Ras/MAPK signaling. Current studies are exploring whether mutations may be more common at recurrence and whether epigenetic regulation of transcription and genomic organization, which has recently been reported to be involved in the medulloblastoma, may be playing similar roles in NB.

Molecular Factors and Expression Signatures

Because recurrent mutations are not frequent in NB, the identification of genes and signaling pathways with altered expression have also been used to discover additional prognostic factors and therapeutic targets involved in NB differentiation, apoptosis, drug resistance, angiogenesis, metastasis, and inflammation. Extensive reviews of these molecular factors have been the subject of several recent reviews, and a subset of the most well studied are included later.

Neurotrophin signaling has central roles in normal neuronal cell development, and the clinical and biological roles of TRK receptors (NTRK1, 2, 3 encoding TrkA, B, C) and their ligands (NGF, BDNF, and NT-3) have been extensively studied in NB (reviewed in Ref. ). TrkA expression is highest in tumors with favorable biological characteristics and outcomes, and TrkA induces apoptosis and/or differentiation in vitro. TrkA signaling has been implicated in mediating spontaneous regression that is often observed in infants with localized or stage 4S disease. In contrast, TrkB has pro-proliferative and migratory properties, enhances chemoresistance, and is highly expressed in biologically unfavorable MYCNA NB. Although the TrkB inhibitor lestaurtinib did not show efficacy in a phase 1 trial, trks and proteins involved in neural crest development and differentiation pathways may still represent potential therapeutic targets.

Disruption of proteins involved in apoptotic pathways, including multidrug-resistance proteins, such as MDR-1, bcl-2 family proteins, caspase-8, mTOR/PI3 kinase, and TP53/HDM2, have also been shown to play important roles in NB initiation and progression. There are many ongoing pre-clinical studies to determine the ability to pharmacologically target these pathways. Many genes involved in NB, including caspase 8 and the RASSF1A tumor suppressor, are inactivated by the promoter hypermethylation, which contributes to resistance to apoptosis induced by many therapies. Demethylating agents, such as decitabine, have been tested in phase I studies. Enhanced angiogenesis and high expression of proangiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor are associated with more aggressive NB tumors; early phase clinical trials of drugs that block these pathways have been completed.

Rather than focusing on specific candidate genes, several investigators have identified multigene expression profiles that predict outcome and may lead to further refinement of risk categories. One large study demonstrated that the expression of 59 genes was an independent predictor of outcome, even after controlling for currently used risk factors, with an odds ratio of 19.3 for OS and 3.96 for progression-free survival. Additional retrospective studies have identified other multigene classifiers (ranging from 3 to >50 genes). Although most of these signatures have not been studied in specific NB risk groups, Asgharzadah and collegues recently demonstrated that a 14-gene classifier can be used to specifically identify subsets of HR patients with the worst prognosis. Many of these signatures include genes implicated in NB pathogenesis, neural development, and inflammation/immune response. Recent reports also demonstrate prognostic profiles of microRNAs, small 22 to 25 nucleotide RNAs that inhibit protein translation or target mRNA degradation (reviewed in Ref. ).