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Current Pediatric Reviews, 2018, 14, 73-90 REVIEW ARTICLE

ISSN: 1573-3963 eISSN: 1875-6336

Biological and Genetic Features of Neuroblastoma and their Clinical Importance

BENTHAM SCIENCE

Nevim Aygun*

Department of Medical Biology, Faculty of Medicine, Dokuz Eylul University, Inciralti, Izmir, Turkey

Abstract: Neuroblastoma derived from primitive cells of the sympathetic nervous system typically develops in the adrenal medulla or paraspinal ganglia. Neuroblastoma usually occurs sporadically, but familial cases are also observed. ALK and PHOX2B germline mutations can cause hereditary neuroblastoma, while a common genetic variation in chromosome 6p22 is associated to sporadic neuroblastoma. However, the aetiology of sporadic neuroblastoma is not exactly known. This embryonic malignancy generally represents the second most common solid tumour after central nervous system tumours throughout the world in childhood. Neuroblastoma is a complex disease that A R T I C L E H I S T O R Y has different clinical courses, from metastatic spread to spontaneous regression. Spontaneous regression can occur without therapy in primary or metastatic site. Potential regression mechanisms

Received: August 11, 2017 primarily involve apoptosis, hypermethylation of subtelomeric DNA, immune response and Nerve Revised: January 18, 2018 Growth Factor (NGF) deprivation. Neuroblastoma is a heterogeneous tumour that can show many Accepted: January 23, 2018 different chromosomal abnormalities; e.g. MYCN amplification, 1p deletion, unbalanced translocations involving chromosome 17, aneuploidies and Loss of Heterozygosity (LOH) events. DOI: 10.2174/1573396314666180129101627 Tyrosine kinase receptors TrkA, TrkB and TrkC, their ligands NGF, Brain-derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT-3), and Aurora Kinase A (AURKA) play a regulatory role in differentiation, apoptosis, cell proliferation, tumourigenesis, angiogenesis or metastasis of neuroblastoma. TrkA expression is associated with differentiation or regression, depending on presence or absence of NGF, whereas TrkB and BDNF are mostly expressed in aggressive neuroblastomas with MYCN amplification. MYCN is amplified in 18-38% of neuroblastoma cases. MYCN amplification mechanism remains to be completely clarified. This paper reviews the biological/genetic features and their clinical importance in neuroblastoma. Keywords: Neuroblastoma, biological and genetic features, clinical importance, genomic signature, chromosomal abnormalities, cell signalling pathways, cell behaviour, clinical behaviour.

1. INTRODUCTION prognosis [11-13]. However, neuroblastoma patients can show different clinical, biological and prognostic Neuroblastoma probably derives from primitive characteristics according to the location of their primary sympathetic neural precursor cells of the peripheral nervous tumour that may arise in the adrenal, abdominal/ system [1]. The majority of these tumours develop in the retroperitoneal, neck, thoracic, pelvic or another site [14]. adrenal medulla, while the remaining ones arise in the This enigmatic tumour can metastasize to one or more paraspinal sympathetic ganglia of the neck, chest, abdomen distant sites from a primary location in the patients with or pelvis [1, 2]. Neuroblastoma usually occurs sporadically, stage 4 or 4S [15], but sometimes the metastatic disease can but familial cases with a prevalence of about 1-2% are also result in spontaneous regression or differentiation, even in reported [3]. Neuroblastoma is the most frequent embryonal cases who do not receive any specific treatment [16]. cancer among children particularly before 5 years of age [4, 5]. This embryonic malignancy generally represents the Using the International Neuroblastoma Staging System second most common solid tumour after central nervous (INSS), neuroblastoma tumours are classified as stages 1, system tumours among children under 15 years of age 2A, 2B, 3, 4 and 4S based on multiple criteria such as degree around the world [5-10]. of surgical excision of primary tumour, lymph node involvement, dissemination to distant organs, degree of bone Infants aged less than 18 months at diagnosis with non- marrow involvement and the age of infant [17]. In addition, a MYCN-amplified neuroblastoma often have a better new classification system called the International

Neuroblastoma Risk Group (INRG) was developed for *Address Correspondence to this author at the Ozkarakaya Avenue, accomplishing the pretreatment staging and risk assessment No. 12/2, 35320, Narlidere, Izmir, Turkey; Tel: of neuroblastoma tumours. By this INRG Staging System Fax: +90-232-238-56-42; E-mail: [email protected] (INRGSS), neuroblastoma tumours are classified as stages

L1, L2, M and MS based on clinical criteria and image- some replication-based mechanisms, the underlying defined risk factors [18]. Stage MS also contains the patients mechanism of MYCN gene amplification remains unclear with stage 3 primary tumours infiltrating the midline, which exactly, as reviewed in [32]. This structural chromosomal are not included within 4S group, although they had a abnormality is significantly associated with age 18 months, limited dissemination. On the other hand, MS group includes stage 4, adrenal and nonthoracic tumour sites, high LDH and the children aged less than 18 months with limited ferritin levels, unfavourable histology, diploidy, 1p deletion, dissemination, whereas 4S group had been limited to infants 17q gain, high Mitosis-karyorrhexis Index (MKI) and younger than 1 year of age [17, 18]. undifferentiated/poorly differentiated grade in neuroblastoma. In addition, MYCN amplification is the most Spontaneous regression that can be observed in many strongly associated with lung metastasis, following with types of cancer is defined as remission or disappearance of a tumour mass without any treatment in primary and/or bone marrow, bone and skin metastases, respectively [33]. metastatic sites, as reviewed in [16, 19, 20]. Spontaneous The frequency of a segmental genomic profile that regression can occur more frequently in children aged less consists of chromosome 1p deletion, 11q deletion and/or 17q than 365 days with stage 1-3 and stage 4S neuroblastoma gain is significantly higher in stage 4 disease and in patients without MYCN amplification [21, 22]. However, a study older than 18 months in neuroblastoma without MYCN reported that an infant with stage 4 neuroblastoma exhibited amplification. A segmental genomic type is also associated a spontaneous regression of tumour at the metastatic site with a higher risk of relapse in the neuroblastoma patients. including meningeal metastasis after gross resection of the 11q deletion, 17q gain, a segmental genomic type and primary tumour [23]. diploidy are significantly associated with decreased Event- free Survival (EFS) and Overall Survival (OS) [34]. TrkA (NTRK1) expression in neuroblastoma tumours contributes to favourable prognosis. TrkA receptor promotes Aneuploidy with numerical chromosomal aberrations in cell differentiation in the presence of NGF ligand in neuroblastoma is a prominent characteristic of a subgroup of neuroblastoma, while it leads to spontaneous regression in infants aged less than 18 months with MYCN amplification the absence of NGF in the microenvironment [24]. A and Uniparental Disomy (UPD). This subgroup that is well dependence receptor UNC5D that is directly targeted by p53 responded to therapy harbours a heterogeneous MYCN interacts with E2F1 after NGF withdrawal, triggering amplification along with UPD11 and carries none or a few transcription of proapoptotic genes and thus mediates the Segmental Chromosomal Aberrations (SCAs), however, programmed cell death in neuroblastoma cells, suggesting diploid/tetraploid tumours that are often associated with that UNC5D involved in the induction of apoptosis caused tumour progression, dissemination and relapse are by NGF deprivation during spontaneous regression of predominant in non-MYCN-amplified group of patients > 18 neuroblastoma [25]. months and display many SCAs including concomitant 11q deletions [35]. Full-length TrkB and BDNF are both expressed in neuroblastoma, particularly in aggressive tumours with Loss of Heterozygosity (LOH) events at chromosomal MYCN amplification [26]. BDNF treatment in TrkB- regions such as 1p, 3p, 4p, 11q and 14q in neuroblastoma expressing neuroblastoma cells increases the cell migration usually occurs due to reduction in one allele, indicating a and invasion. In addition, TrkB-expressing xenograft hemizygous deletion mechanism that involves neuroblastoma tumours in mice demonstrate distant haploinsufficiency or inactivation by intrachromosomal metastases, indicating that TrkB/BDNF expression increased deletions, mutations or epigenetic repression in the retained the metastasis in vitro and in vivo. PI3K and MAPK alleles; however, copy-neutral events with normal copy pathways mediate the action of TrkB/BDNF on number observed at 11p, which are thought to be formed neuroblastoma cell migration and invasion during metastasis through UPD, may be a cause for LOH [36]. [27]. However, disruption of neurotrophin-3 (NT-3), a TrkC To explore molecular mechanisms underlying the cellular ligand, triggers cell death in malignant human neuroblasts and clinical behaviours occurring throughout the progression and inhibits tumour growth and metastasis in the xenograft of neuroblastoma, this review will focus on the chromosomal model of both chick and mouse [28]. abnormalities and cellular signalling pathways involving LIN28B-RAN-AURKA signalling network in apoptosis, differentiation, spontaneous regression, neuroblastoma drives oncogenesis and promotes proliferation and metastasis. tumourigenesis. AURKA depletion decreases MYCN expression, whereas high AURKA expression is associated 2. CLINICAL, BIOLOGICAL, GENETIC AND BIO- with reduced overall survival [29]. AURKA induces the cell CHEMICAL FACTORS AFFECTING PROGNOSIS OF proliferation, anchorage-independent cell growth and VEGF- NEUROBLASTOMA mediated angiogenesis through regulation of nuclear translocation of MYCN in neuroblastoma cells [30]. 2.1. Short Historical Background MYCN amplification is a poor prognostic factor in Neuroblastoma was first described by Dr. Rudolf neuroblastoma. Stage 4 tumours that harbour MYCN Virchow in 1864, as a glioma tumour developing in the amplification display progression most rapidly, suggesting abdominal cavity in a child [37]. The characteristic features that MYCN amplification may play a key role in bad and origin of neuroblastoma were examined in detail by Dr. prognosis in aggressive neuroblastomas [31]. Although it James Homer Wright in 1910, based on his personal was proposed that MYCN oncogene might be amplified via Biological, Genetic and Clinical Characteristics of Neuroblastoma Current Pediatric Reviews, 2018, Vol. 14, No. 2 75 observations in the histopathological specimens of multiple of paediatric cancers in Low- and Middle-income Countries cases and findings of previously reported cases. (LMIC) that have a population-based registry, however, its true incidence in other most LMIC is unknown. He was the first person to name this mysterious tumour as neurocytoma or neuroblastoma after he observed that This discrepancy between HIC and LMIC for the more or less undifferentiated nerve cells or neurocytes or incidence of neuroblastoma may be due to reasons such as neuroblasts were the essential cells of this tumour. He late or suboptimal diagnosis, insufficiency in pathologic determined that the ball-like neuroblasts termed rosettes evaluation with immunohistochemistry, advanced imaging surrounding the fibrils that arise in the adrenal medulla have techniques and available resources, and unavailable a morphology similar to those that arise in the sympathetic infrastructure in LMIC, as reviewed in [39]. nervous system, suggesting that neuroblastoma is a tumour originated from the migrated primitive nerve cells [38]. 2.3. Clinical, Biological, Genetic and Biochemical He had also seen that neuroblastoma is not a very rare Characteristics tumour, and thus he estimated that the tumours of this type Low to high-risk groups in neuroblastoma show different may constitute an important group among the gliomas, clinical, biological and genetic characteristics (Table 1). sarcomas, lymphomas or lymphosarcomas. Very Low-risk (VLR) patients with small tumour masses have an excellent favourable outcome with 4-year OS > 95% 2.2. Incidence without any surgical or medical intervention. Low-risk (LR) patients with INSS 1 and 2 tumours have also good Neuroblastoma is the most common extracranial solid prognosis with 3-year OS > 95%. Surgical resection and tumour in High-income Countries (HIC), and it accounts for following observations are sufficient for these asymptomatic 6-9.7% (ratio of annual rates per million population) of all patients whose tumours are largely (> 50%) resected, cancers observed in children younger than 15 years of age [7, whereas the patients with non-MYCN-amplified tumours in 9, 10]. On the other hand, neuroblastoma accounts for 1-3% the Intermediate-risk (IR) group are treated with moderate-

Table 1. Clinical, biological and genetic characteristics, and biochemical parameters that affect prognosis in neuroblastoma*.

INSS 1 2 3 4 4S

VL (<6m), L, I or I or H, H (18m) L, I or Risk group L H (MNA) H (MNA) or I (nMNA) H (MNA) Prognosis

Age 18 months + + W

MYCN amplification + + W

MYCN mRNA expression Low/- Low/- High High Low/- -/B (nMNA)

W or MYCN protein expression Rare Rare High High Rare B (nMNA)

TrkA expression High High Low Low High B

Full-length TrkB/BDNF High High W expression

TrkC expression High High Low Low High B

Near-triploidy + + + B

Diploidy/Near-diploidy + + W

1p36 LOH + W

11q LOH + W

17q gain + W

LDH + + W

Ferritin + + W

Urinary VMA, normal + + -

NSE + + W

*All data presented here were obtained from the references [11, 16, 26, 31, 33, 39-53]. Abbreviations: B, Better; H, high; I, Intermediate; L, Low; LDH, Lactate Dehydrogenase; LOH, Loss of Heterozygosity; M, Month; MNA, MYCN- amplified; nMNA, non-MYCN-amplified; NSE, Neuron-specific Enolase; VL, Very Low; VMA, Vanillylmandelic Acid; W, Worse. 76 Current Pediatric Reviews, 2018, Vol. 14, No. 2 Nevim Aygun intensity chemotherapy. High-risk (HR) neuroblastoma can with 1p36 LOH or 11q LOH have decreased 3-year EFS and be treated with multimodal therapies consisting of intensive OS rates, compared to groups without 1p36 LOH or 11q induction chemotherapy, radiotherapy, myeloablative LOH [49]. The gain of 17q is also significantly associated chemotherapy with autologous hematopoietic stem cell with stage 4 disease. In addition, 17q gain is associated with rescue and treatment of Minimal Residual Disease (MRD) age 1 year, 1p deletion, MYCN amplification and [39]. diploidy/tetraploidy. 17q gain is considered an important poor prognostic factor in neuroblastoma [50]. Patients aged < 18 months in neuroblastoma show significantly higher 3-year OS and EFS rates, compared with Prognostic significance of biochemical parameters such patients aged > 18 months [11]. Age 18 months is as LDH, ferritin, Vanillylmandelic Acid (VMA) and significantly associated with MYCN amplification in Neuron-specific Enolase (NSE) in neuroblastoma was also neuroblastoma [33]. Neuroblastoma patients with stages 3 investigated. High LDH level is associated with MYCN and 4 generally have a poor prognosis [40] and MYCN amplification, high histologic grade and the decreased amplification is associated with advanced-stage disease and survival rate [33, 51]. In addition, MYCN amplification is poor prognosis [31, 41]. However, the prevalence of MYCN significantly more often detected in children with normal amplification in neuroblastoma patients with low stages 1 VMA urinary excretion, high ferritin or NSE serum level. and 2 is around 3%, and the patients in this subgroup have High-level of ferritin or NSE significantly decreases the less favourable prognosis than those with non-MYCN- survival rate, whereas urinary VMA does not affect survival amplified tumours of INSS 1 and 2 [42]. [52, 53]. The mRNA expression of MYCN is usually low or absent in low to intermediate-risk groups, whereas the most 3. AETIOLOGY AND TUMOURAL BEHAVIOUR OF neuroblastoma tumours of a high-risk group that generally NEUROBLASTOMA harbours MYCN amplification demonstrate higher MYCN expression [43]. The mRNA level of MYCN is also lower in 3.1. The Genetic and Biological Aetiology of Hereditary stage 4S neuroblastoma tumours without MYCN and Sporadic Neuroblastoma amplification compared to MYCN-amplified tumours [44]. Although neuroblastoma is the most common embryonic MYCN protein is rarely expressed in stages 1, 2 and 4S, malignancy diagnosed during the first year of life [5], only a whereas MYCN overexpression is more frequently observed few inherited mutations that predispose to this mysterious in stages 3 and 4, showing that its expression can be a tumour were reported in a limited number of neuroblastoma predictor for worse prognosis [45]. families. Three germline missense mutations (G1128A, The high mRNA expression of TrkA is significantly R1192P and R1275Q) identified in the tyrosine kinase associated with stages 1, 2 and 4S tumours, as well as domain of protein product encoded by the Anaplastic younger age (< 1 year). In addition, high-level expression of Lymphoma Kinase (ALK) oncogene were shown to co- both MYCN and TrkA in neuroblastoma patients without segregate with the disease in eight separate families who had MYCN amplification is associated with a higher survival rate individuals affected by neuroblastoma, demonstrating that than that with low expression of both [46]. Neuroblastoma these heritable mutations can lead to familial neuroblastoma patients who have tumours with positive TrkA transcript [54]. Two germline missense mutations (R100L and R141G) expression show a better OS compared to that of those with located in the homeodomain of the paired-like homeobox 2B negative TrkA expression, regardless of the risk group [43]. (PHOX2B) in a familial case and in a patient with Full-length TrkB and BDNF are both expressed generally in neuroblastoma were also reported, suggesting that these the aggressive neuroblastomas with MYCN amplification germline heterozygous mutations can predispose to [16, 26]. TrkC is significantly more expressed in the lower hereditary neuroblastoma [55]. stages 1, 2 and 4S than in the advanced stages 3 and 4. The On the other side, a homozygosity (GG) for the at-risk G expression of TrkC is positively correlated with survival, allele representing a Single-nucleotide Polymorphism (SNP) whereas its expression is negatively correlated with MYCN at chromosome 6p22, containing the predicted gene amplification [47]. FLJ22536 that encodes a potential protein with an Epidermal DNA ploidy level affects the survival rates in Growth Factor (EGF)-like domain, increases the likelihood neuroblastoma. Near-triploid tumours are significantly of the development of sporadic neuroblastoma [56]. associated with patients under 1 year of age, stages 1, 2 and Depletion of a short isoform (CASC15-S) of CASC15 that is 4S and no MYCN amplification. Patients with near-triploid a tumour suppressor long noncoding RNA at 6p22 enhances tumours have a 2-year disease-free survival rate of 94% the proliferation and invasiveness of neuroblastoma cells, compared to those of 45% and 11% in the patients with suggesting that a SNP in a tumour suppressor gene at 6p22 diploid or near-diploid tumours, without and with MYCN locus may increase the susceptibility to neuroblastoma [57]. amplification, respectively [48]. 1p36 LOH and 11q LOH In addition, it has recently been reported that enforced are significantly more often observed in stage 4 than in MYCN expression in p53-compromised neural crest cells stages 1, 2, 3 and 4S. 1p36 LOH is significantly associated isolated from mouse embryo generated a neuroblastoma with MYCN amplification and diploid tumours, whereas 11q tumour in immune-deficient mice, showing structural LOH is correlated with non-MYCN-amplified tumours. In chromosomal abnormalities syntenic to segmental addition, neuroblastoma tumours with 1p36 LOH or 11q aberrations such as 17q gain, 2p gain and 1p36 LOH closely LOH display the associations with age > 1 year, related to human MYCN-amplified neuroblastoma [58]. unfavourable histopathology and high-risk group. Patients However, the question about how early-stage neuroblastoma Biological, Genetic and Clinical Characteristics of Neuroblastoma Current Pediatric Reviews, 2018, Vol. 14, No. 2 77 tumours with low mRNA and protein expression of the the abdominal/retroperitoneal (64% ± 1.1% and 72% ± MYCN gene (Table 1) could be formed, remains unanswered. 1.1%), neck (79% ± 2.8% and 90% ± 2.2%), thoracic (80% ± 1.2% and 88% ± 1.0%) and pelvic (81% ± 2.6% and 91% ± 3.2. The Primary Sites of Neuroblastoma Tumour 2.0%), respectively (P < 0.001) [14]. Neuroblastoma generally arises in the adrenal medulla, In another study, abdominal tumours compared to extra- however, the paraspinal sympathetic ganglia in the neck, abdominal ones are significantly associated with the high- chest, abdomen or pelvis can also be a primary tumour site risk group, disseminated stages 4 and 4S, MYCN [1, 2]. Among the neuroblastoma patients, the most common amplification, elevated levels of LDH, ferritin and NSE, and primary tumour site is adrenal (47%), followed by the decreased 5-year EFS rate [59]. abdominal/retroperitoneal (24%), thoracic (15%), pelvic (3%) and neck (3%) [14]. 3.3. Metastasis in Neuroblastoma Adrenal primary tumour site is significantly associated The most common sites of metastases that are observed with age 18 months at diagnosis, INSS stage 4 disease, in children with stage 4 neuroblastoma are bone marrow, MYCN amplification, diploidy/hypodiploidy, 1p LOH, 17q bone, distant lymph nodes and liver, whereas lung, Central gain, 11q aberration, elevated serum ferritin ( 92 ng/mL) Nervous System (CNS) and skin are much less common sites and LDH ( 587 U/L) levels, unfavourable INPC pathology [14, 33]. Many metastasis-related genes that involve in and high MKI. The unadjusted 5-year EFS (56% ± 0.8%) different cellular pathways in neuroblastoma have been and OS (62% ± 0.8%) rates of infants who have a primary determined (Table 2). tumour in the adrenal are significantly worse than those in

Table 2. Genes related to metastasis in neuroblastoma.

Preferential Gene Locusa Classb Pathway Function in Metastasis References Metastasis

Inducer of proliferation, migration and HIF-1 14q23.2 TF SHH Lymph node Chen et al. [62], invasiveness

Facilitator of glucose transport (upregulated GLUT1 1p34.2 GlcT HSP Gut and liver Herrmann et al. [63] in hypoxia)

Reversible hydration of C0 (upregulated in CA9 9p13.3 TMP HSP Gut and liver 2 Herrmann et al. [63] hypoxia)

Inducer of angiogenesis (upregulated in VEGF 6p21.1 GF HSP Gut and liver Herrmann et al. [63] hypoxia)

Gut, liver and Inducer of apoptosis Herrmann et al. [63] CASP8 2q33.1 PE HSP bone marrow (downregulated in hypoxia) and Teitz et al. [64]

Tumour suppressor (downregulated in DCC 18q21.2 TMR HSP Gut and liver Herrmann et al. [63] hypoxia)

MMP9 20q13.12 ZDEP HSP Gut and liver ECM protease (upregulated in hypoxia) Herrmann et al. [63]

ADAMTS1 21q21.3 METP HSP Gut and liver ECM protease (upregulated in hypoxia) Herrmann et al. [63]

TIMP1 Xp11.3 ECM protease inhibitors (upregulated in METPI HSP Gut and liver Herrmann et al. [63] TIMP2 17q25.3 hypoxia)

CDH1 16q22.1 CDCAP HSP Gut and liver Cell adhesion (downregulated in hypoxia) Herrmann et al. [63]

TIMP inhibitor, CDH1 downregulation TWIST1 7p21.1 TF HSP Gut and liver Herrmann et al. [63] (upregulated in hypoxia)

20q13.13 Gut, liver and Herrmann et al. [63] SNAI1 SNAI2 TF HSP APOP EMT and invasion (upregulated in hypoxia) 8q11.21 pararenal area and Vitali et al. [65]

MMP2 16q12.2 ZDEP HSP Gut and liver ECM protease (upregulated in hypoxia) Herrmann et al. [63]

VCAM1 1p21.2 TIMP HSP Gut and liver Cell-cell adhesion (upregulated in hypoxia) Herrmann et al. [63]

ICAM1 19p13.2 TMP HSP Gut and liver Cell adhesion (upregulated in hypoxia) Herrmann et al. [63] (Table 2) Contd… 78 Current Pediatric Reviews, 2018, Vol. 14, No. 2 Nevim Aygun

Preferential Gene Locusa Classb Pathway Function in Metastasis References Metastasis

5q11.2 ITGA1 3q21.2 Cell-matrix adhesion Herrmann et al. ITG5 ITG3 CSR HSP Gut and liver 17q21.32 [63] ITGV (upregulated in hypoxia) 2q32.1

CTNNA1 5q31.2 CAP HSP Gut and liver Adhesion molecule (upregulated in hypoxia) Herrmann et al. [63]

Anti-adhesive molecule (downregulated in VCAN 5q14.2-3 EMP HSP Gut and liver Herrmann et al. [63] hypoxia)

Lung and lymph It enhances tumour cell growth, proliferation, 45A ncRNA ? ncRNA MIG Penna et al. [66, 67] nodes migration and metastatic potential

Its overexpression upregulates focal adhesion miR-659-3p 22q13.1 miRNA FAP Bone marrow Stigliani et al. [68] pathway and represses metastasis

Its overexpression suppresses cell growth, miR-337-3p 14q32.2 miRNA EPIGP Lung Xiang et al. [69] invasion and angiogenesis

Its overexpression suppresses cell growth, miR-338-3p 17q25.3 miRNA PTEN/ AKT - Chen et al. [70] proliferation, migration and invasion

Its overexpression promotes cell growth, miR-558 2p22.3 miRNA EPIGP Lung Qu et al. [71] invasion and angiogenesis

1q22 Its overexpression suppresses cell growth, miR-9 (1-3) 5q14.3 miRNA ANG Lung Zhang et al. [72] migration, invasion and angiogenesis 15q26.1

HOXD-AS1 ANG It controls RA-induced cell differentiation, Yarmishyn et al. 2q31.1 lncRNA - (HAGLR) JAK/ STAT angiogenesis and inflammation [73]

Lung and bone PHOX2B 4p13 TF EPIGP It suppresses the metastasis Naftali et al. [74] marrow

ANG It suppresses the growth, invasion and ITLN1 1q23.3 Lectin Lung Li et al. [75] PI3K/AKT metastasis

It suppresses the growth, invasion, FOXD3 1p31.3 TF ANG Lung Li et al. [76] angiogenesis and metastasis

It enhances the cell growth, proliferation, AKT2 19q13.2 PK ANG Liver Qiao et al. [77] migration, invasion and angiogenesis

FAK It increases cell survival, migration and 8q24.3 NRPTK - Liver Megison et al. [78] (PTK2) invasion

MEN2B RET 10q11.21 RTPK MAPK/JNK Lung It enhances cell growth and metastasis Marshall et al. [79]

1p36.12/ Ambrosio et al. LSD1/MYCN MAOX/TF EMTP - They increase the migration and invasion 2p24.3 [80] a Chromosomal locations were obtained from the HUGO gene nomenclature committee (HGNC) website (http://www.genenames.org/) [60]. b Class of gene products was obtained from GeneCards (Human gene database, http://www.genecards.org/) [61]. Abbreviations: ANG, Angiogenesis; APOP, Apoptosis; CAP, Cell Adhesion Protein; CDCAP, Calcium- Dependent Cell Adhesion Protein; CSR, Cell-surface Receptor; ECM, Extracellular Matrix; EMP, Extracellular Matrix Protein; EMT, Epithelial to Mesenchymal Transition; EMTP, epithelial to mesenchymal transition pathway; EPIGP, Epigenetic Pathway; FAP, Focal Adhesion Pathway; GF, Growth Factor; GlcT, Glucose Transporter; HSP, Hypoxia Signalling Pathway; lncRNA, long noncoding RNA; MAOX, Monoamine Oxidase; MAPK/JNK, Mitogen-activated Protein Kinase/Jun N-terminal Kinase; METP, Metalloproteinase; METPI, Metalloproteinase Inhibitor; MIG, Migration; miRNA, microRNA; ncRNA, noncoding RNA; NRPTK, non-receptor Protein Tyrosine Kinase; PE, Proenzyme; PI3K/AKT, phosphoinositide-3-kinase/AKT; PK, Protein Kinase; RA, Retinoic Acid; RTPK, Receptor Tyrosine Protein Kinase; SHH, Sonic Hedgehog; TF, Transcription Factor; TIMP, Type I Membran Protein; TMP, Transmembran Protein; TMR, Transmembran Receptor; ZDEP, Zinc-Dependent Endopeptidase.

Hypoxia-inducible Factor-1 (HIF-1) promotes the differentiation in neuroblastoma tumours [62]. Most proliferation, migration and invasiveness of neuroblastoma neuroblastoma primary tumours and neuroblastoma cell lines cells under hypoxia in vitro and enhances the growth and express a high-level of SHH, PTCH1, SMO and GLI2 angiogenesis of xenograft tumours in nude mice in vivo components of SHH signalling pathway [81]. The through regulation of the sonic hedgehog (SHH) signalling Transforming Growth Factor (TGF)-1 induces Epithelial to pathway. The expression of SHH and GLI1 is associated Mesenchymal Transition (EMT) in neuroblastoma cells and with advanced stage, lymph node metastasis and poor increases GLI1 and GLI2 expressions in the cells undergoing Biological, Genetic and Clinical Characteristics of Neuroblastoma Current Pediatric Reviews, 2018, Vol. 14, No. 2 79

EMT, leading to cell migration. During EMT, the expression downregulation of PHOX2B in lung micrometastatic of mesenchymal marker -SMA is upregulated, conversely, neuroblastoma cells results in a significant increase of that of epithelial cell marker E-cadherin is downregulated tyrosine hydroxylase expression [74]. The positive [82]. Slug (SNAI2) also induces EMT. SNAI2 and -SMA expression of tyrosine hydroxylase in peripheral blood at (ACTA2) are both overexpressed in neuroblastoma [83]. diagnosis in neuroblastoma patients is significantly LMO4 is an essential cofactor of SNAI2 that mediates associated with stage 4 metastatic disease and its higher cadherin repression during EMT and its activity is required expression predicts a worse outcome [86]. for neuroblastoma cell invasiveness [84]. On the other side, AKT2, focal adhesion kinase (FAK) Hypoxic conditions (1% oxygen for 3 days) cause cell and multiple endocrine neoplasia 2B (MEN2B) RET migration towards blood vessels for intravasation, slow function as enhancers of neuroblastoma metastasis [77-79] down cell velocity within blood vessels for extravasation and (Table 2 and Fig. 1). An effector of PI3K/AKT pathway, promote cell proliferation in primary and secondary sites in AKT2 kinase induces liver metastasis. It regulates MYCN neuroblastoma cells. During this metastatic progression, expression and involves VEGF secretion in neuroblastoma hypoxia leads to the activation or inactivation of expression cells [77]. FAK that is a non-receptor protein tyrosine kinase of many metastasis-related genes such as GLUT1, VEGF, also increases liver metastasis in vivo [78]. MEN2B RET MMP2, MMP9, SNAI1, SNAI2, VCAM, ICAM, ITGA1, increases lung metastases in vivo and activates the CASP8, DCC, CDH1 and VCAN [63], see Table 2 and Fig. 1. MAPK/JNK pathway [79]. In addition, MYCN and the Lysine-specific Demethylase 1 (LSD1) inhibit the expression Noncoding RNAs (ncRNAs) play varied roles such as of MYCN downstream-regulated gene 1 (NDRG1) that is a regulating gene expression, catalyzing DNA synthesis, metastasis suppressor, providing control of EMT and tumour protecting genome particularly through avoiding telomere metastasis in neuroblastoma [80]. shortening, and modulating chromatin looping and histone modifications in numerous biological processes including transcription, RNA processing, translation, replication, 3.4. Spontaneous Regression in Neuroblastoma telomere maintenance and chromatin remodelling. Majority On the other hand, stage 4S neuroblastoma tumours that of ncRNAs like snoRNPs, telomerase, microRNAs are prone to spontaneous regression more frequently (miRNAs) and long ncRNAs compose of RNA-protein metastasize to the liver (80.2%), bone marrow (34.6%) and complexes and sequence-specific bind to other nucleic acids skin (13.6) than lymph node (8.6%) and adrenal (6.2%) [15]. or proteins in the nucleus, cytoplasm or both of eukaryotic Spontaneous regression is known to involve different cells [85]. In neuroblastoma cells, multiple noncoding RNAs biological processes, even if its potential formation act as a regulator in one or more of cell growth, proliferation, mechanism remains to be fully solved (Fig. 2). differentiation, inflammation, adhesion, migration, invasion or angiogenesis pathways during metastasis [66-73]. The TrkA receptor expression that is a favourable prognostic miR-9, miR-337-3p, miR-338-3p and miR-659-3p suppress factor in neuroblastoma causes the differentiation in presence the metastasis in neuroblastoma, whereas miR-558 and 45A of its ligand NGF in the microenvironment, but it leads to ncRNA promote it (Table 2 and Fig. 1). spontaneous regression in the absence of NGF [24]. A high PES1 expression that is associated with advanced stage 4 PHOX2B, intelectin 1 (ITLN1) and FOXD3 that all and MYCN amplification induces tumour cell growth and attenuate the metastatic colonies in the lung function as inhibits apoptosis in neuroblastoma cells. However, NGF- suppressors of metastasis in neuroblastoma [74-76]. The

AKT2, miR-558 VEGF miR-337-3p, miR-9, ITLN1, FOXD3

Tumour cells Angiogenesis 45A ncRNA Lymph node metastasis

a SHH pathway Hypoxia SHH Lymph node (low O ) GLI1 2 HIF-1 Growth and Efferent Afferent TGF-b1,Slug EMT proliferation lymphatic FAP lymphatic vessel

GLI1/2 , a-SMA miR-659-3p vessel CASP8 DCC Invasion LMO4, PREX2a, AKT2, FAK, LSD1/MYCN LSD1/MYCN, FAK, AKT2,45A ncRNA Migration Blood MMP2/9 Intravasation vessel miR-338-3p

Apoptosis Tumour FOXD3, ITLN1 ITGA1 Extravasation suppression Proliferation Metastatic site

FOXD3, ITLN1, PHOX2B, miR-9, miR-337-3p Lung metastasis 45A ncRNA, miR-558, MEN2B RET Bone marrow CASP8, PHOX2B, miR-659-3p metastasis

Liver metastasis AKT2, FAK

Fig. (1). Numerous metastasis-related genes function as an enhancer or suppressor of metastasis during cell growth, proliferation, angiogenesis, EMT, migration, invasion, intravasation and extravasation in disseminated neuroblastoma [62-84]. A number of genes are more often associated with metastasis to a specific site such as lung, bone marrow, lymph node or liver. Abbreviations: EMT, Epithelial to Mesenchymal Transition; FAP, Focal Adhesion Pathway. 80 Current Pediatric Reviews, 2018, Vol. 14, No. 2 Nevim Aygun

Apolar NGF NGF Monopolar ...... Bipolar Ras-MAPK pathway ...... TrkA receptor TrkA Differentiated cells

Neuroblasts Decreased DNA repair JNK pathway c-Jun PES1 PES1 RAD51

NGF deprivation KIF1Bb Nuclear accumulation of DHX9 XAF1

Caspases 2/3 Proapoptotic UNC5D UnlCD + E2F1 UnlCD/E2F1 complex Cell proliferation gene expression Apoptosis T Cell-Mediated Immunity IL-2 and IL-27 CD8+ T cells Caspase activation CTL responses IFN- No NGF ligand Y Tumour HOXC9 NGF mass deprivation Tumour mass Which pathway? Spontaneous regression Epigenetic Control

TrkA receptor TrkA Hypermethylated Hypomethylated PVRL3 3 MCM2/7 EIF5A C9orf3 SLC9A5 EPHB1 G-quadruplex 3-overhang ALDOA KCNQ5 5 JSRP1 RUNX1T1 Telomeres Hypermethylation FHOD1 FOXP2 Telomerase GPN3 PTEN OR activity Promoter +1 TERT gene

Telomere shortening

Fig. (2). Potential mechanisms underlying spontaneous regression in neuroblastoma. A neuroblastoma tumour is mostly composed of apolar, monopolar and bipolar cell types, along with a few multipolar cells [87, 88]. TrkA receptor mediates differentiation or tumour regression based on the presence or absence of NGF ligand, respectively. Cellular signalling pathways involving differentiation, apoptosis and impaired DNA repair, NGF deprivation, adaptive T cell-mediated immunity, telomere shortening and epigenetic control contribute to spontaneous regression in neuroblastoma [24, 25, 89-96]. induced differentiation decreases PES1 expression, homeobox gene HOXC9 causes a significant decrease in cell suggesting that stage 4S tumours with lower PES1 viability in neuroblastoma cell lines and represses tumour expression are prone to differentiation or spontaneous growth in neuroblastoma xenografts. HOXC9 re-expression regression [89]. In addition, the ablation of PES1 in also triggers an intrinsic apoptotic pathway involving the colorectal cancer cells treated with etoposide resulted in activation of caspases [93], suggesting an association decreased DNA repair involving RAD51, indicating that between apoptosis and neuroblastoma spontaneous repressing PES1 can inhibit proliferation of cancer cells [90]. regression. The PES1 expression is upregulated by JNK signalling On the other side, the promoter of TERT gene encoding pathway in colon cancer [91]. the catalytic subunit of telomerase, which is located on A kinesin tumour suppressor KIF1B that is located at subtelomere of chromosome 5p, is hypermethylated in stage 1p36.22 [60] interacts with RNA helicase A (DHX9) and 4S neuroblastoma tumours compared to stage 4. This leads to its nuclear accumulation when NGF becomes epigenetic control causes lower expression level of TERT limiting. DHX9 accumulation stimulates proapoptotic XIAP- gene in stage 4S, suggesting that shortening telomeres may Associated Factor 1 (XAF1), resulting in apoptosis in counteract tumour cell immortalization and thus contribute neuroblastoma cells. In addition, 1p36-deleted to spontaneous regression in stage 4S neuroblastoma. neuroblastomas demonstrate the loss of KIF1B expression Additionally, the promoter of the genes such as MCM2/7, and impaired nuclear localization of DHX9 [92]. In addition, EIF5A, SLC9A5 and FHOD1 is hypermethylated in stage 4S a dependence receptor UNC5D is cleaved by caspases 2/3 tumours, whereas the promoter of numerous genes such as after NGF withdrawal. As a cofactor of E2F1, caspase- EPHB1, KCNQ5, PTEN, C9orf3 and PVRL3 is released UnICD fragment interacts with E2F1 at the hypomethylated [94]. Telomestatin that is an agent promoter of E2F1-target genes in the nucleus and both stabilizing G-quadruplex structure limits the cellular lifespan transactivate the transcription of proapoptotic target genes, of neuroblastoma cells to inhibit telomerase activity, leading to programmed cell death in neuroblastoma cells resulting in telomere shortening, growth arrest and apoptosis [25]. [95]. The elevated HOXC9 expression is associated with Combined treatment of IL-2 and IL-27 induces potent spontaneous regression in neuroblastoma. Re-expression of antitumour activity to enhance CD8+ T cells, tumour-specific Biological, Genetic and Clinical Characteristics of Neuroblastoma Current Pediatric Reviews, 2018, Vol. 14, No. 2 81

Cytotoxic T Lymphocyte (CTL) responses and IFN- structures regarding both dmin and hsr forms in two production in metastatic neuroblastoma and triggers subclones (one with the only hsr at 5q and other with the complete tumour regression and long-term survival in mice only dmin) of STA-NB-10 cell line with a deletion at 2p24.3 bearing widely metastatic neuroblastoma tumours, indicating in only one of two chromosomes 2 were identical and whose contribution of adaptive T cell-mediated immunity to fusion junctions were mediated by Nonhomologous end spontaneous regression of disseminated neuroblastoma Joining (NHEJ) [102], supporting episome model of gene tumour [96]. amplification proposed by Carroll et al. [103]. According to this model, after a submicroscopic chromosomal region 4. CHROMOSOMAL ABNORMALITIES AND containing a replication origin along with adjacent genes was CELLULAR SIGNALLING PATHWAYS IN deleted by a recombination event, autonomously replicating NEUROBLASTOMA submicroscopic precursors called episomes generate larger dmins, later which can integrate into a new chromosomal MYCN 4.1. Amplification and Its Potential Formation site. Mechanisms Of somatically acquired rearrangements involving mostly MYCN amplification, which has been reported in 18-38% intrachromosomal tandem duplications and amplicons in two of primary tumours as well as multiple cell lines in lung cancer cell lines NCI-H2171 (small-cell lung cancer neuroblastoma [41, 97-101], is an important poor prognostic cell line) and NCI-H1770 (neuroendocrine lung cancer cell factor for this embryonic malignancy [31]. MYCN oncogene line), 53% contain a microhomology between fused ends, is amplified either at its resident site 2p24 on the short arm ranging from 1 to 10 bp in length. NCI-H1770 cell line of chromosome 2, which can be observed in numerous harbours an hsr containing MYCN amplification in direct neuroblastoma cell lines including CHP134, KPNYS, IMR- orientation on chromosome 2. In addition, this amplicon 5, GOTO and NB17 along with many other solid tumours contains the acquired rearrangements occurring in both such as carcinomas of lung, adrenal, breast and oesophagus inverted and noninverted orientations, suggesting either that or germ cell tumours [32], or at another chromosome, as this hsr containing MYCN amplification has undergone identified in Kelly, IMR32 and MHH-NB-11 neuroblastoma further rearrangements after insertion of a dmin through cell lines (unpublished data). Amplified segments in their episome model or that extrachromosomal dmin involved in chromosomal site are manifested as homogeneously staining an error-prone replication [104]. regions (hsr) (Fig. 3a-c, unpublished data). However, MYCN amplification can also occur as extrachromosomal double Recently, I investigated the microhomology level minutes (dmin), as detected in SIMA neuroblastoma cell line between the sequences of both 150 bp spanning 5' and 3' (Fig. 3d, unpublished data). boundaries of amplicon units containing MYCN amplification that are located at the short arm of A study demonstrates that eight neuroblastoma (7 STA- chromosome 2 in 14 neuroblastoma cell lines and 42 other NBs and 1 SK-N-BE) and two small cell lung carcinoma primary solid tumours [32], whose genomic data were (GLC8 and GLC14) cell lines contained the amplicons obtained from the catalogue of somatic mutations in cancer showing MYCN amplification as hsr or dmin, the amplicon

a Kelly b IMR32 c MHH-NB-11 d SIMA (hsr) (hsr) (hsr) (dmin)

chr13 hsr

hsr chr17 hsr chr1

dmin

Fig. (3). Metaphase and interphase areas display hsrs and dmin in neuroblastoma cell lines (unpublished data, Aygun N and Altungoz O). The hsrs containing MYCN amplification are located on chromosomes 17, 1 and 13 in Kelly, IMR32 and MHH-NB-11 cells, respectively. The dmins are seen as extrachromosomal structures in SIMA cells. Fluorescence in situ Hybridization (FISH) probe: MYCN gene (2p24)/Chromosome 2 Alpha-Satellite (red/green, Qbiogene, cat. no., PONC0224). (The color version of the figure is available in the elec- tronic copy of the article) 82 Current Pediatric Reviews, 2018, Vol. 14, No. 2 Nevim Aygun

(COSMIC database, http://cancer.sanger.ac.uk/cosmic/) at the genomic sites up to 8-kb away from the breakage site [105]. The results showed a microhomology with mean 5.18 in Saccharomyces cerevisiae [113]. A LIR with 111-bp stem bp ranging from 2 to 14 bp between the boundary sequences and 24-bp internal spacer can extrude a hairpin structure on of the amplicon units. Microhomologies between the both leading and lagging strand templates during replication. sequences spanning 5' and 3' boundaries are formed between In addition, a cruciform can lead to replication fork stalling perfect or imperfect repeats particularly in longer [114]. Fork stalling or collapse may also be induced by a microhomologies at the junction or nearby location [32]. single-strand DNA break [115]. Taken together, the results Imperfect microhomology at the junctions is a characteristic suggest that LIRs can trigger MYCN amplification possibly feature of template-switching events occurring during DNA through either replication fork stalling or break-induced replication. Amplified DNAs that are induced under stress in replication in a manner dependent on microhomology during E. coli are composed of tandem direct repeats of 7-32 kb, DNA synthesis. I have also previously demonstrated that which are flanked by short G-rich repeats showing a LIRs are significantly associated with the breakpoint regions microhomology of 4 to 15 bp, some of them are imperfect of human gross gene deletions, suggesting that LIRs can repeats. In this study, a novel long-distance template- cause gene deletions via induction of DNA breaks during switching model mechanism (later called the fork stalling replication, which are likely to involve an end-joining repair and template switching, FoSTeS) that uses microhomology mechanism [116, 32]. for gene amplification has been first proposed [106]. For the formation of MYCN amplification, the Canonical NHEJ uses either no microhomology or mechanisms involving unscheduled DNA replication, terminal (junctional) microhomology of 1-4 nucleotides recombination or extra replication followed by excision of [107]. Flanking microhomologies along with junctional ones the amplified DNA, dmin formation and later in situ are also detected in template-switching events that lead to amplification after integration of dmin into a chromosomal complex rearrangements in yeast. It was suggested that site, resulting in hsr, were also proposed [117, 118]. Other strand invasion is more likely to be influenced by these general replication-based models proposed for gene microhomologies that flank the breakpoint than the amplification such as DNA re-replication, double rolling- junctional ones [108]. In addition, the majority of TMPRSS2- circle replication and FoSTeS can also be considered for the ERG gene rearrangements in prostate cancer occur at or near mechanism underlying MYCN amplification, as reviewed in the microhomology or involve insertion of one or more base [32]. pairs [109]. On the other hand, a 2.8 Mb non-fragile region 4.2. 1p36 Deletion and Its Involved Potential Candidate containing MYCN gene locates between two common Fragile Tumour Suppressor Genes Sites (cFS) called FRA2Ctel and FRA2Ccen at 2p24.3 and MYCN amplification shows significant correlations with 2p24.2, respectively. 56.5% of MYCN amplicons that were high MKI and undifferentiated/poorly differentiated tumours identified in neuroblastoma cell lines and primary tumours in neuroblastoma [33, 98]. In addition, both MYCN cluster in FRA2C, suggesting that unbroken DNA secondary amplification and 1p36 deletion demonstrate a significant structures extruding at FRA2C during DNA synthesis could association with high tumour vascularity in neuroblastoma trigger extra replication rounds in this region, causing high- [119], suggesting that MYCN-involved oncogenic level MYCN amplification [110]. transformation and an impaired tumour suppressive function In addition, I identified the long inverted repeats (LIRs) promote angiogenesis and metastasis in neuroblastoma (Fig. in a human genomic DNA segment lying between 2p25.3 4). Candidate loci for potential tumour suppressor gene(s) and 2p24.3 [32], using the inverted repeat finder (IRF) involved in deletions of the short arm of chromosome 1 in software (https://tandem.bu.edu/cgi-bin/irdb/irdb.exe) [111]. neuroblastoma have been identified in primary tumour The vast majority (75.38%) of LIRs were distributed along tissues and many cell lines (Table 3). the second half of this segment, containing MYCN locus at Among these loci, it is seen that 1p36 is a commonly 2p24.3. 18.85% of LIRs with stem identity 70% in this deleted region in neuroblastoma, as indicated by LOH region had stem length 20 bp, stem identity 85% and a studies (Table 3). To explore the role of deletions of the loop length of 0-2 kb [32], which may be potentially short arm of chromosome 1 in the development of recombinogenic [112]. The results also demonstrated that neuroblastoma, this commonly deleted 1p36 locus was mean LIR number was significantly higher in both 5' and 3' subject to further analyses. A study group reported that about rearrangement boundaries of the amplicon units containing 15% of MYCN single copy neuroblastomas display 1p MYCN gene in neuroblastoma cell lines and other primary deletions, whose SRO of 47 centirays (cR) maps to 1p36.3 solid tumours, whose boundary positions were obtained from and furthermore that these lost alleles are of preferential COSMIC database [105], compared to control group, maternal origin, suggesting that 1p36.3 may be an imprinted respectively (P < 0.05; P < 0.01) [32]. Additionally, many locus. Group also showed that the lost alleles in MYCN- LIRs were identified both inside and outside the amplicon amplified neuroblastoma tumours with much larger deletions units, suggesting that a hairpin and a cruciform extruded by extending from 1p36 to the telomere are of random parental these LIRs, respectively, can cause replication fork stalling origin [131, 134]. However, another study reported that the during DNA synthesis [32]. lost alleles at 1p36.3 locus identified in neuroblastoma Inverted repeats can promote chromosomal fragility and tumours without MYCN amplification did not demonstrate a induce mutagenesis via formation of increased DNA breaks preferential parental origin [135].

Biological, Genetic and Clinical Characteristics of Neuroblastoma Current Pediatric Reviews, 2018, Vol. 14, No. 2 83

Table 3. Candidate loci for the potential 1p tumour suppressor gene(s) of neuroblastoma.

SRO With Specimen Method Year Reference (Case Number) MNA (Case Number)

Primary tumour 1p36.1-3 (13) + (8) Southern blotting (45) 1989 Fong et al. [120] Cell line (2)

(1p35-36.1)-pter (9) + (9) Somatic cell hybrid mapping Cell line (16) 1995 Cheng et al. [121] 1p36.23-33 or no del (7) -

PCR-based DNA Primary tumour Martinsson et al. 1p36.22-33 (12) + (10) 1995 Polymorphisms (46) [122]

1p36.2-3 [stage 4 (40)] + S4 (26) 1p22 [stage 4 (41)] Primary tumour 1p36.2-3 [stage 1-3 (14)] + S1-3 (3) Allelic analysis with microsatellite markers 2000 Mora et al. [123] (120) 1p22 [stage 1-3 (10)] 1p32 and MycL [4S (3)] - 4S

Primary tumour 1p36.1-pter (32) + (32) Southern blot analysis with polymorphic markers (171) 2000 Spieker et al. [124] 1p36.3 (?) - Cell line (25)

+ (2) PCR-based LOH analysis with polymorphic markers 1p36.2-3 (2) (HDR) Cell line (2) 2000 Ohira et al. [125] [105, 126]

LOH analysis with polymorphic markers Primary tumour 1p36.3 (135, primary ? (503) 2001 White et al. [127] tumour) Cell line (46)

1p32-34 (17) + (13) LOH analysis with polymorphic markers Primary tumour 2001 Hiyama et al. [128] 1p36.1-2 (20) + (1) (92) 1p36.3 (6) -

Multiplex PCR-based LOH analysis with polymorphic Primary tumour 1p36.3 (93) + (48) 2001 Maris et al. [129] markers (288)

PCR-based LOH analysis with polymorphic markers Primary tumour 1p36.3 (15) + (2) 2001 Bauer et al. [130] (49)

1p36.1-pter (31) + (31) Primary tumour Southern blot analysis with polymorphic markers 2001 Caron et al. [131] 1p36.3 (22) - (205)

LOH analysis with polymorphic markers Primary tumour 1p36.2-3 (75) + (56) 2001 Hogarty et al. [132] (75)

LOH analysis with polymorphic markers Primary tumour 1p36.3 (224) ? (737) 2005 White et al. [133] Cell lines (46)

Abbreviations: HDR, Homozygously Deleted Region; LOH, Loss of Heterozygosity; MNA, MYCN Amplification; SRO, Smallest Region of Overlap.

On the other side, a Homozygously Deleted Region amplified tumours [136]. In addition, KIF1B is (HDR) of 500 kb at 1p36.2-3 in two neuroblastoma cell lines hemizygously deleted in 84% of MYCN-amplified primary NB-1 and NB-C201 was identified (Table 3). The expression neuroblastomas. The decreased expression of KIF1B is not of HDNB1/UFD2, DFF45/ICAD and KIAA0591F/KIF1B due to promoter hypermethylation. Low expression of genes located in this region was significantly higher in KIF1B increases cell proliferation and promotes tumour favourable stage 1 tumours than in MYCN-amplified formation in nude mice, whereas its overexpression induces unfavourable tumours [125]. KIF1B is also silenced in 1p36 an apoptotic cell death involving caspases in a p53- hemizygous-deleted neuroblastomas. Low KIF1B independent manner (Figs. 2 and 4), suggesting that KIF1B expression predicts a worse outcome in non-MYCN- 84 Current Pediatric Reviews, 2018, Vol. 14, No. 2 Nevim Aygun

Chromothripsis 2p24 3p14-25 RASSF1A TAp73 E2F1 MYCN amplification deletion PUMA E2F1 MOAP-1 11q23.3 17q gain TSLC1 MYCN MYCN/LSD1 deletion ncRAN

Bax MYCN TERT Tumour cells Angiogenesis NDRG1 17q Growth FOXD3 gain BIRC5 RA Bmi1 VEGF MMP14, AURKA, MDM2 s i Apoptosis Proliferation s

p E2F1 i r

h Caspase- Migration t CAMTA1 RA o dependent p19 miR-337-3p m

o Invasion r h

. .

. C

. TAp73 . miR-34a .

. .

. . .

. . . .

. . 14q32 . . .

...... PI3K/MAPK . .

. . . . .

...... Blood . .

......

. .

. pRb . . . .

. . deletion Cdc42 1p36 KIF1Bb vessel Promoter E2F hypermethylation deletion BDNF BDNF CAMTA1 Proliferation Cdc42 Nm23 Differentiated cells CHD5 RUNX3 CASZ1 Metastatic site (1p36) MYCN-induced miRNAs nm23-H1 Epigenetic receptor TrkB Bmi1 NGF NGF silencing 17q gain 18q21 DCC 11p gain BDNF deletion (Hypoxia) MYCN E2F1 AHR EZH2 TrkA receptor TrkA

Fig. (4). Chromosomal abnormalities and signalling networks that control cell growth, proliferation, angiogenesis, metastasis, apoptosis and differentiation pathways in neuroblastoma. Structural abnormalities of chromosomes 1, 2, 3, 11, 14, 17 and 18 involving allelic loss of 1p, 3p, 11q, 14q and 18q, amplification of 2p24 including MYCN gene, and gain of 11p and 17q affect signal networks related to many oncogenes and potential tumour suppressor genes such as MYCN, BIRC5, KIF1B, CHD5, p73, RASSF1A, TSLC1 and DCC [26-30, 63, 69, 76, 80, 136-179]. Abbreviations: miRNAs, micro RNAs; RA, retinoic acid. may act as a haploinsufficient tumour suppressor gene in regulator of miR-34a [142]. The miR-34a from 1p36.22 [60] neuroblastoma [137]. was shown to be a potent tumour suppressor in vivo in neuroblastoma [143]. The expression of other candidate tumour suppressor CHD5 located at 1p36.31 is very low or absent in The role of another candidate tumour suppressor neuroblastoma cell lines and its promoter is highly CAMTA1 was also investigated in primary neuroblastomas. methylated in NLF and IMR5 cells with hemizygous 1p Low expression of CAMTA1 was significantly associated deletion and MYCN amplification, suggesting that CHD5 with 1p deletion, MYCN amplification, advanced stage and functions as a tumour suppressor regulated through an poor outcome [144]. CAMTA1 slows cell proliferation, epigenetic mechanism in neuroblastoma. The high CHD5 suppresses tumour growth in vivo and induces neurite-like expression is strongly associated with younger age, lower processes and markers of neuronal differentiation in stage, no MYCN amplification, hyperdiploidy, favourable neuroblastoma cells [145]. CASZ1, other candidate tumour histopathology and normal 11q status [138]. Expression of suppressor from 1p36, induces cell differentiation, enhances the remaining allele of CHD5 deleted at 1p36 may also be cell adhesion and inhibits cell migration in neuroblastoma controlled via another epigenetic mechanism involving cells. CASZ1 also inhibits tumour growth in vivo, causing a MYCN-driven miRNAs. Recently, the miR-211, -17, -93, - suppression of tumourigenicity. In addition, CASZ1- 20b, -106b, -204 and -3666 have been shown to expressing tumours show an increase in the expression of downregulate CHD5 expression in neuroblastoma cells. NGFR (p75NTR) and TrkA [146]. CASZ1 suppresses cell Among these miRNAs, miR-17, -93, -106b and -20b are proliferation to inhibit E2F transcriptional activity via upregulated by MYCN [139], indicating the cross-talk restoring pRb activity in neuroblastoma cells [147]. EZH2 between MYCN oncogene and a possible 1p36 tumour induces the H3K27me3-mediated epigenetic gene silencing suppressor gene in neuroblastoma cells. The CHD5 of CASZ1 in neuroblastoma cells [148]. expression is upregulated by NGF only in TrkA receptor- expressing neuroblastoma cells, resulting in neuronal 4.3. 11q LOH differentiation [140]. 11q loss, an important prognostic marker, defines a The expression of p73 gene located at 1p36.3 is much biologically distinct group of non-MYCN-amplified tumours, less often detected in primary neuroblastomas than in which can demonstrate a metastatic aggressive growth in primary colon and breast cancers. p73 LOH shows a neuroblastoma. This chromosomal loss frequently occurs significant association with sporadic neuroblastomas, MYCN along with loss of chromosomes 3p, 4p and/or 14q, and is amplification and advanced stage tumours. The mutations of significantly associated with poor outcome [149]. 11q LOH p73 gene in neuroblastoma were rare, suggesting that p73 is generally associated with 17q gain and 3p LOH in primary may not function as a classical tumour suppressor, neuroblastoma tumours with single-copy MYCN gene, conforming to the Knudson's hypothesis [141]. Both whereas MYCN amplification is mostly associated with 1p TAp73 and TAp73 isoforms of TAp73 protein induce deletion and 17q gain [150]. In addition, 1p22 and 1p31-p34 miR-34a transcription, which in turn mediates RA-induced LOH are observed along with 11q and/or 14q LOH in non- neuroblastoma cell differentiation, indicating that TAp73 is a MYCN-amplified stage 4 tumours [151]. Biological, Genetic and Clinical Characteristics of Neuroblastoma Current Pediatric Reviews, 2018, Vol. 14, No. 2 85

A chromosomal site 11q23.3 is commonly deleted in candidate locus for tumour suppressor gene(s) in neuroblastoma tumours with 11q LOH. This allelic loss is neuroblastoma patients. inversely correlated to MYCN amplification [152]. TSLC1/IGSF4/CADM1, a candidate tumour suppressor gene, 4.5. 17q Gain is mapped to chromosome 11q23 and whose expression is downregulated in unfavourable neuroblastomas. TSLC1 On the other hand, overexpressed nm23-H1 from decreases cell proliferation in SH-SY5Y neuroblastoma cell 17q21.33 [60] inhibits Cdc42-induced differentiation line, suggesting that this gene may function as a tumour through binding to Cdc42. Both nm23-H1 and Cdc42 are the suppressor in neuroblastoma [153]. downstream targets of MYCN. In neuroblastoma cells, MYCN upregulates nm23-H1, whereas it downregulates MYCN directly activates Bmi1 gene, resulting in the Cdc42 [165]. acceleration of proliferation and inhibition of differentiation in NB cells. Bmi1 represses the tumour suppressors KIF1B Survivin (BIRC5) gene from 17q25.3 [60] is expressed in and TSLC1 through a Polycomb group gene-mediated many primary neuroblastomas and neuroblastoma cell lines. epigenetic chromosome modification [154], suggesting that Its high-level expression is significantly associated with the remaining allele of TSLC1 was inactivated via an advanced stage and low-level of TrkA expression. Survivin epigenetic modification in neuroblastoma. Bmi1 is also transfection into neuroblastoma cell line CHP134 inhibits the upregulated by E2F1 in neuroblastoma cells [155]. RA-induced apoptosis possibly via inhibition of caspase-3 activation [166]. 4.4. The Other LOHs Involving Chromosomes 14q, 3p The expression of ncRAN from 17q25.1 is upregulated in and 18q advanced neuroblastomas with 17q gain. The ectopic expression of ncRAN induces cell transformation in NIH3T3 On the other hand, a LOH study that was performed in cells, whereas its knock-down inhibits cell growth in SH- primary neuroblastomas using the microsatellite markers SY5Y cells, suggesting that this non-coding RNA can act showed a smallest common region of allelic loss at 14q32, like an oncogene in aggressive neuroblastomas [167]. suggesting the presence of one or more tumour suppressor genes in this chromosome band [156]. A microRNA miR- 337-3p at band 14q32.2 represses the expression of MMP14, 4.6. Aneuploidy causing decreased expression level of VEGF in Among Whole Chromosome Aneuploidies (WCAs), neuroblastoma cell lines. In addition, its overexpression trisomy of chromosomes 6-9, 12, 13, 17, 18, 20 and 21 as suppresses the growth, invasion, angiogenesis and metastasis well as monosomy of chromosomes 3, 4, 9-11, 15, 17, 19, 22 of neuroblastoma cells in vitro and in vivo [69], see also and X compared to other chromosomes are more frequently Table 2 and Figs. 1 and 4. observed in neuroblastoma, according to data obtained A consensus region of allelic losses identified at from the Mitelman database (https://cgap.nci.nih.gov/ chromosome 3p in neuroblastoma tumours is located in a Chromosomes/ Mitelman/) [168]. large region between 3p14.3 and 3p25.3 [157]. The Favourable neuroblastoma tumours (including mostly promoters of RASSF1A from 3p21.3 and CASP8 are both stages 1, 2 and 4S) with predominantly near-triploidy and hypermethylated in a significant number of neuroblastomas, numerical aberrations demonstrate a typical gain of suggesting that RASSF1A may act as a tumour suppressor chromosomes 6, 7 and 17, and loss of chromosomes 3, 4, 11 gene in neuroblastoma [158]. The complete methylation of and 14 [150]. In the high-risk group without MYCN RASSF1A promoter was significantly more prevalent in amplification in stage 4 neuroblastoma, the patients with MYCN-amplified tumours than in MYCN-single copy WCA 2 show significantly higher survival probability neuroblastomas [159]. RASSF1A demonstrates the apoptotic compared to ones with WCA <2. Patients in this group activity either via Modulator of Apoptosis-1 (MOAP-1) or usually have whole chromosome gains mostly in TAp73 in multiple tumour cells including neuroblastoma cell chromosomes 7, 12 and 17 as well as allelic loss of 11q and lines [160-162]. 3p, and 17q gain [169]. DCC tumour suppressor gene from 18q21.2 [60] displays absent or reduced mRNA expression in many neuroblastoma 4.7. Chromothripsis cell lines and primary tumours, some of them include 18q Chromothripsis, a massive genomic rearrangement LOH. Reduced expression of DCC is significantly associated acquired in a single catastrophic event observed in at least 2- with advanced stage neuroblastomas, suggesting that the 3% of all cancers, particularly in bone cancers (25%) [170], downregulation of DCC expression is a poor prognostic was also identified in 18% of primary tumours with stages 3 factor in neuroblastoma [163]. In addition, DCC expression and 4 in neuroblastoma. Chromothripsis-related structural is downregulated during hypoxia, when the neuroblastoma aberrations are associated with amplification of MYCN or cells metastasize [63]. CDK4 genes and 1p LOH [171], suggesting that In addition, we recently identified a novel deleted region chromothripsis represses neuroblastoma cell differentiation on the long arm segment q25-q41 of chromosome 1 in Kelly via allelic loss of some potential 1p36 tumour suppressor human neuroblastoma cell line [164]. This locus is likely to genes involving induction of differentiation (Fig. 4). contain one or more tumour suppressor genes important for Chromothripsis also leads to structural rearrangements of neuroblastoma progression. Further studies are necessary to TERT, resulting in a significant increase in telomere length investigate the loss of heterozygosity at 1q25-q41 as another in high-stage neuroblastomas [172], suggesting that 86 Current Pediatric Reviews, 2018, Vol. 14, No. 2 Nevim Aygun chromothripsis contributes to cell immortalization with After MDM2 was translocated from the nucleus to the unlimited proliferative capacity. cytoplasm during hypoxia, the C-terminal RING domain of MDM2 protein binds to an AU-rich sequence within the 3' 4.8. Signalling Networks Controlling Cellular Processes untranslated region (3' UTR) of VEGF mRNA, resulting in an increase of VEGF mRNA stability and translation. TrkB and BDNF are both expressed in many aggressive MDM2 promotes tumour growth and metastasis through primary neuroblastomas [26] and play an important role in regulation of VEGF expression in neuroblastoma cells [180]. neuroblastoma cell migration and invasion during metastasis via PI3K and MAPK pathways [27]. TrkA and its ligand CONCLUSION NGF involve cell differentiation and spontaneous regression, as reviewed in the section entitled 'Aetiology and tumoural Neuroblastoma is the most common cancer diagnosed behaviour of neuroblastoma' (Fig. 2). Upregulation of NT-3 during the first year of life. Many clinical, biological, genetic prevents TrkC-induced cell death and promotes survival of and biochemical factors such as age 18 months, MYCN neuroblastoma cells [28]. amplification, 1p deletion, 17q gain, 11q LOH, diploidy, TrkB/BDNF expression, and high LDH and ferritin levels are BDNF gene is located at 11p14.1 [60]. The gain of 11p associated with worse prognosis. Metastatic stage 4 occurs more frequently in 11q-deleted neuroblastomas. The neuroblastomas with MYCN amplification, 1p deletion, 17q SRO of 11p gain is between chromosome bands 11p11.2 and gain, diploidy, TrkB/BDNF expression and high LDH level 11p14 [173], suggesting that 11p gain leads to the overexpression of BDNF. A candidate neuroblastoma show more aggressive tumour behaviour, further declining survival rates. However, 11q LOH, 17q gain and 3p LOH tumour suppressor RUNX3 from 1p36.11 [148, 60] constitute a distinct group within non-MYCN-amplified abrogates TrkB expression in neuroblastoma cell lines [174]. tumours, which is associated with poor outcome. LIN28B-RAN-AURKA signalling promotes On the other side, numerous genes such as HIF-1, tumourigenesis in neuroblastoma. Depletion of AURKA causes decreased MYCN expression; conversely, high VEGF, CASP8, MMP2/9, noncoding RNAs, PHOX2B, AKT2 and LSD1/MYCN act as either an inducer or a AURKA expression is associated with poor outcome [29]. In suppressor of neuroblastoma metastasis. NGF/TrkA addition, AURKA regulates the nuclear translocation of signalling, various other signalling pathways involving MYCN and induces cell growth and proliferation as well as apoptosis, differentiation and impaired DNA repair, telomere VEGF-mediated angiogenesis [30]. STK15 (AURKA) that shortening, adaptive T cell-mediated immunity and encodes a centrosome-associated kinase, was reported to be amplified and overexpressed in multiple human tumour cell epigenetic control contribute to spontaneous regression of neuroblastoma. Taken together, it is evident that types including neuroblastoma. In addition, it was shown chromosomal abnormalities and both oncogenes and tumour that AURKA overexpression leads to centrosome suppressor genes, along with clinical characteristics, which amplification, aberrant chromosome segregation, affect the progression of neuroblastoma, are necessary to be aneuploidy, chromosomal instability and transformation in evaluated together to develop new treatment strategies for mammalian cells [175]. the patients, especially in aggressive neuroblastomas. During EMT, MYCN and LSD1 colocalize at the promoter of NDRG1 tumour suppressor gene and repress its CONSENT FOR PUBLICATION transcription, causing an increase in motility and invasiveness of neuroblastoma cells [80], see Figs. 1 and 4, Not applicable. and Table 2. The expression of VEGF and MMP9 is downregulated by NDRG1. FOXD3 tumour suppressor CONFLICT OF INTEREST facilitates NDRG1 transcription through binding to its The author declares no conflict of interest, financial or promoter [76]. otherwise. E2F1, E2F2 and E2F3 transcription factors induce MYCN transcription via binding to its proximal promoter in vivo ACKNOWLEDGEMENTS specifically in neuroblastoma cells [176]. In addition, E2F1 Declared none. induces the transcription of p19 that is a CDK4/6 inhibitor in SH-SY5Y neuroblastoma cell line, leading to repression of cell proliferation [177]. The overexpression of AHR REFERENCES downregulates the expression of MYCN and E2F1 genes in [1] Brodeur GM. Neuroblastoma: biological insights into a clinical neuroblastoma cells, promoting cell differentiation through enigma. Nat Rev Cancer 2003; 3(3): 203-16. regulation of E2F1 [178]. [2] Maris JM. Recent advances in neuroblastoma. N Engl J Med 2010; 362(23): 2202-11. In response to nutlin-3 treatment in a p53-null and [3] Malis J. 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