Neuroblastoma is a childhood malignant form of cancer that originates from neuroblasts, and affects children under five years of age and rarely occurs in adults. It is a tumor that arises from neural crest cells and mainly affects ssthe adrenal medulla. There is remarkable genetic heterogeneity and chromosomal abnormality that leads to this malignant pediatric tumor.

Purpose: This article is mostly focused on genetic and molecular abnormalities that lead to neuroblastoma. Heterogeneity of MYCN gene and over-expression of MYCN gene as well as translocations or mutation in gene like ALK (anaplastic lymphoma kinase), PHOX2b gene, rearrangements of ATRX and TERT and also chromosomal deletions and gain that are associated with neuroblastoma that are also discussed. Moreover, in this article associated target therapy against specific genes is also discussed.

Neuroblastoma is a type of tumor of the peripheral nervous system that primarily affects children. It arises from immature nerve cells that are known as neuroblasts. In normal conditions neural crest cells migrate from the dorsal neural tube, unfortunately in some cases there are defects in neural crest migration, maturation or differentiation that can lead to development of neuroblastoma [1]. These cells are commonly found on adrenal gland, neck, chest, abdomen or pelvis. So, these areas are commonly affected in neuroblastoma. Neural crest cells maturation, migration and differentiation is regulated by genes [2] thus abnormalities in genes like overexpression of genes lead to tumors. However, neuroblastoma is a type of tumor that highly depends on several driver and suppressor genes. In the genetic view neuroblastoma associated with MYCN gene overexpression, Somatic and germline mutation like ALK [3], and PHOX2B gene [4], mutation of ATRX gene [5], rearrangement of TERT [6] these are predominant in high risk neuroblastoma.

Moreover, chromosomal alterations include deletions of chromosomes 1p,3p,4p and also 11p and gains of 1p,2p and 17q chromosomes associated with high risk neuroblastoma [7]. MYCN genes have groups including N-myc that have additionally 2 sub-groups (C-myc and L-myc ). These MYC proteins are regulators of cell fate and also interact with transcription factors. That transcription factor regulates neural crest cells differentiation, migration and maturation so in MYCN gene overexpression this factor leads to malignant tumors like neuroblastoma and often with poor survival. MYCN gene is one of the prognostic markers, presence of this marker indicates poor prognosis of neuroblastoma [2]. ALK has been classified in a superfamily of tyrosine kinase. The ALK gene is located on the 2p23 chromosome that encodes receptor tyrosine kinase and somatic mutations have been confirmed in the tyrosine kinase domain in that ALK gene is present that is associated with primary type of neuroblastoma. So currently for therapy of ALK positive neuroblastoma molecular inhibitors against ALK can be given [8] 

CD44 is a particularly transmembrane adhesive protein involved in tumor metastasis and progression. It regulates cellular growth, motility as well as differentiation. It works as a main receptor for the ligand hyaluronic acid and as other extracellular components [9]. CD44 is found as a good prognosis of neuroblastoma tumor and also it is associated with glial differentiation [9]. Overview of MYCN gene in neuroblastoma The MYCN gene is found at the terminal end of the short arm of chromosome 2. MYCN gene belongs to a very small family, MYCN(N-Myc) gene includes 2 more genes C-Myc and L-Myc. N-Myc is mainly associated for initiating cancer like neuroblastoma. N-Myc works as a cancer stem cell factor in the development of neural crest cells like neuroblasts and C-Myc is a pluripotent stem cell in embryonal development, it promotes development of pluripotent stem cell from ectoderm in embryonal period then it further develops into neural crest cells and MYCN gene then plays main role in differentiation of those neural crest cells. However, in this time overexpression of MYCN gene leads to tumor development [2]. MYCN alone plays multiple roles in development of neuroblastoma, it can activate transcriptions of genes that can be involved in self renewal, angiogenesis, survival and proliferation. It also suppresses the expression of genes that promote cell differentiation, moreover it also activates apoptosis via p53 pathway [10]. Study showed that in neuroblastoma with MYCN gene overexpression p53 protein is a direct target of the MYCN gene and p53 protein is expressed at much higher levels in poor differentiated or undifferentiated types of neuroblastoma [11]. MYCN amplification causes down regulation of TP53 inducible protein 1 and upregulation of SKP2 and CDK4 that show an inability to arrest in G1 phase of cell cycle in responset irradiation and proliferation of cell continues, MYCN gene also directly represses the activity of anti-proliferative proteins [10].

Structure of MYCN gene:

The MYCN gene is made of 464 amino acids and it has so many functional domains. The MYCN gene contains a basic helix-loop-helix-leucine zipper that plays an oncogenic role in cancer, it is responsible for physical interaction and binding to DNA sequence 5′-CACGTG-3′ and that is termed as E-box [12], [13]. This family basic helix-loop-helix-leucine zipper has transcription factors that play a role in cellular proliferation, differentiation, apoptosis and tumorogenesis[11]. Gene transcription of MYCN is regulated by enhancers and promoters, enhancers play an important role in cell type specific gene expression and they are misregulated in neuroblastoma like cancers. CDK1 is a stability regulator protein of MYCN gene, in normal condition CDK1 phosphorylates the MYCN at S62 in G- phase of cell cycle, other stabilizers are AURKA (AA17-43; 61-89), FBXW7(AA61-89) [12].

Mechanism of action:

Molecular mechanisms: The expression of MYCN gene is whether activated or not at DNA, mRNA and proteins is defined by several factors including secondary form of DNA structure, enhances and transcription factors [14]. MYCN gene Through gene amplification can cause proto-oncogene overexpression. This type of process involves formation of 

extrachromosomal DNA that leads to increase of gene copy number, it is done by DNA recombination and replication [15].

So abnormal N-Myc is based on gene amplification, enhanced translation and transcription [14].

Metabolic reprogramming:

Part of the MYCN gene c-myc regulates amino acid transporter ASCT2 and c-myc induces metabolic reprogramming in CD44 positive cancer cells [13]. The study also showed that the MYCN gene amplification type of neuroblastoma is dependent on ASCT2 for maintaining sufficient levels of glutamate to activate TCA cycle.

Further, MYCN amplification type of neuroblastoma cells needs machinery that keeps metabolic demand of glutathione as is mainly necessary for activation of TCA cycle [13], [16]. Studies showed that the MYCN gene uses aerobic glycolysis for energy in neuroblastoma cells [17], [18]. MYCN also regulates so many cellular processes during development of normal cellular and cancer formation. MYCN increases glycolytic flux and glutaminolysis and that further helps to develop tumorigenesis in cancer like neuroblastoma [19].

P53 in neuroblastoma:

P53 is identified as the saviour of the genome and this p53 gene mutation leads to most malignancy to develop, but in the neuroblastoma mutation of this gene found rarely and surprisingly it accumulates in neuroblastoma [11].

As a prognostic factor:

The MYCN gene has no such reliable antibody that can be used in IHC. We have to detect MYCN amplification at the nucleic level [20]. Survival outcomes in neuroblastoma patients are usually affected by Schwann cell specific types of genes like (CALR, KLF10, UBL3) [21]. Neuroblastoma with MYCN positive expression or overexpression is classified as high-risk and needs a higher dose of chemotherapy than others [20]. High-risk neuroblastoma patients have a survival chance less than 50%, with therapy and chemo they survive less [18].

Targeted therapy options against MYCN gene:

MYCN gene drives the upregulation of ABC transported and also the metabolic enzymes that contribute to chemoresistance, this can be the reason for patients with MYCN-amplified gene neuroblastoma. They often respond poorly to chemotherapy [22].

Against MYCN gene small-molecular inhibitors like PI3K and GSK3 inhibitors have been identified as reduced viability specifically in MYCN-amplified cell line xenografts [23]. These GSK3 inhibitors act on a large scale on neuroblastoma cells and cause cell death [24]. PI3K/AkT/mTOR pathway is activated in most types of the neuroblastoma. It is a pro survival signaling pathway that prompts neuroblastoma cell survival and chemoresistance [25]. PI3K inhibitors alone showed results but cells over time developed clinical resistance, to overcome that study showed that therapy with multidrug is effective. Therapy with targeting PIM, PI3K and mTOR is effective after this therapy tumor showed lots of differentiation and cell death and tumor growth also reduced over time [26]. There is p53-MDM2 pathway where MDM2 inhibits the activity of p53 and blocks the effects of p53 also increases MYCN gene translation and tumorigenesis [25]. This pathway is associated with MYCN gene overexpression [27]. So, small molecular inhibitors that inhibit the p53-MDM2 pathway can be very useful for patients with high-risk MYCN-amplified neuroblastoma [28].

Alteration of ALK gene

ALK (anaplastic lymphoma kinase) is similar to an insulin receptor. It is a receptor of tyrosine kinase and belongs to a superfamily of insulin receptors and contains glycine rich domains. In neuroblastoma gain-of-function mutation in the kinase domain is seen [29]. In 85% of cases mutation is seen on F1174, F1245 and R1275. These mutations are found in both sporadic and familial types of neuroblastoma. Study showed that sometimes these ALK mutations are correlated with MYCN gene [29]. ALK signaling pathway is associated for activating PI3K-AKT, JAKSTAT and MAPK pathways. These pathways are able to cell proliferation, cell survival and some like PI3K-AkT shows chemoresistance also [30]. In neuroblastoma ALK signaling shares similarities with MYCN gene to growth of the tumor development so it is also a high-risk type of neuroblastoma [30]. In formation of neuroblastoma ALK gene abnormalities like mutation, amplification or translocation can cause proliferation of neuroblast cells. It is thought that during cell migration the ALK gene can increase their migration rate. The ALK gene is required only for migration and cell proliferation, however it is not required for differentiation so it only affects migration and proliferation [31].  As a treatment option ALK inhibitors like crizotinib and ceritinib are now developed but the tumor cell can develop clinical resistance and may need multiple therapy options [32].

Mutation and alteration in genes

PHOX2B gene

It is the first gene to be identified in neuroblastoma predisposition, it is believed that this PHOX2B gene encodes a very important transcription factor that is necessary for autonomic nervous system development [31]. High levels of the PHOX2B gene showed that it promotes cell proliferation and growth of neuroblast cells. It shows effects on early stages of neuroblasts growth and is likely associated with poor differentiation of cells [33]. There is BMP signaling that is done by dorsal aorta and it initiates the neural crest cells to express high levels of PHOX2B and that activates some proteins like Phox2A, GATA2 and GATA3 and that further promotes cell differentiation [34]. With mutation of the PHOX2B gene this process gets blocked. The PHOX2B protein is encoded by the PHOX2B gene that is located on chromosome 4p13. The PHOX2B mutation is seen on both familial and sporadic types of neuroblastoma. Study showed that it is found as undifferentiated and poorly differentiated and presents as an optimal diagnostic marker. It is sensitive for diagnosis for neuroblastoma not only on tissue specimens but cytological specimens also [35].  ATRX gene The ATRX gene is located on the long arm of X chromosome and it encodes ATP-dependent helicase SWI/SNF (switch/sucrose non-fermentable) that is chromatin remodeller family. It is involved in many functions like DNA repair, transcription regulation and nucleosome recognition. In the neuroblastoma there is point mutation or frameshift mutation that results in loss of function of the ATRX gene [36]. ATRX is also involved indirectly in inhibiting macroH2A1 deposition, removing R loops and also modifying histones. Some studies showed that 90% of ATRX deletion neuroblastoma also had 11q deletion [5].

Maybe 11q deletion existed before ATRX but there is no data about that. Why this occurs needs further research. MYCN gene amplification with ATRX mutation leads to suppression of the tumor suppressor gene found in neuroblastoma from patients of all types of stages and ages and MYCN and ATRX are incompatible and both leads to DNA replicative stress and ATRX mutation type of neuroblastoma have often worse outcome [19].

TERT gene

Telomerase are located at the end of the chromosomes in eukaryotes. The enzyme telomerase consists of two components, one is TERT it is telomerase reverse transcription protein that is responsible for catalytic activity within cells and other one is TERC that is RNA subunit and works as a template for synthesizing sequences of telomerase [37].  TERT is a reverse transcription that is expressed by stem cells. Its function is to use noncoding RNA templates and telomeric repeats at telomers, that prevents apoptosis mediated by telomers. DNA methylation can inhibit the expression of telomerase and MYCN expressed neuroblast cells express TERT [38]. In neuroblastoma there is induction of TERT gene and activation of ALT pathway is seen and MYCN gene also induces TERT gene and increases its effect in tumor development and combination of this leads to high-risk neuroblastoma with poor outcomes [37]. NALT is alternative lengthening of telomerase that is associated with ATRX mutation and can be seen with TERT gene mutation [36].

BARD1 gene

BARD1 gene mutation is often characterized as a rare type of mutation in neuroblastoma and is a high-risk and predicts worst outcomes. BARD1 gene works as a DNA repair it binds to BRCA1 and that further repairs DNA damage [39]. The BARD1 gene is associated with germline mutation and in neuroblastoma it is rarely found. With repairing of DNA, it is also associated with modulation of chromatin structure, hormone signaling and cell cycle regulation. The full length of the BARD1 gene is also found to be a tumor suppressor gene [40]. MYCN plays a main role in DNA repair and with DNA damage by BARD1 gene neuroblast cells must be synthesizing more MYCN genes [41]. 

NF1 gene

NF1 is a major mediator of peripheral neuronal nerve cells progenitors. It is studied that in MYCN expressed NF1 loss and activation of RAS-MAPK pathway alters neuroblastoma and drives tumorigenesis crazy [42], [43]. Binimetinib is the drug that inhibits MEK that indirectly inhibits the RAS-MAPK pathway that showed good results in NF1 gene mutated neuroblastoma [44]. The main function of the NF1 gene involves RAS proteins and its activation. Neurofibromin is a very long amino acid that GTPase activates proteins and GAP related protein and domains. The RAS proteins are most of the time bound to inactivate form of GDP and in time of activation they can form RAS-GTP and can activate one more pathway PI3K/AKT/mTOR and this pathway has one unique ability to protect the cells from apoptosis [45].

RAS-MAPK pathway alteration in neuroblastoma

The RAS-MAPK pathway is activated in the primary type of neuroblastoma and association of this pathway with neuroblastoma showed poor prognosis and worse outcomes. Mutation in genes like ALK showed activation of this pathway [46]. Although alteration of this pathway frequently occurs in relapsed neuroblastoma tumors [47]. PHOX2B gene also through various mutations found to activate MAPK pathway [48]. NF1 gene mutation found in neuroblastoma that is also associated with activation of the RAS/MAPK pathway [44]. This pathway is a signaling pathway and for cellular function sends different signals and alters cellular function, in dysregulation of this pathway cancerous event occurs. It regulates transcription of many genes for cellular function; it regulates it via phosphorylation [49]. This pathway contains two components RAS and MAPK, p44 is mainly associated with downstream signal cascade of the RAS protein and MAPK (p44/42 mitogen-activated protein kinase) is extracellular signal-regulated protein kinases that is the one that overexpressed in neuroblastoma and found in another type of cancers too [47].

For therapy MEK1/2 inhibitor drug-Binimetinb showed some possibilities. In that study specific MEK1/2 inhibitor drug-Binimetinb was sensitive with some of the cell lines and on that cell death were seen with cell rounding and detachment, however much more cell lines were resistant to treatment and showed no results [44]. Other analogues are Cobimetinib, Selumetinib and Trametinib can be given to inhibit the RAS-MAPK pathway in neuroblastoma [49].

PI3K/AKT/mTOR pathway

The ALK gene is one of the prognostic markers for high-risk neuroblastoma that activates this PI3K/AKT/mTOR pathway [25]. NF1 gene and many other genes can alter and activate this pathway [45]. This pathway prompts the neuroblastoma cell survival and protects them against chemotherapy so it makes tumors even stronger [25]. For targeted therapy against this pathway, multikinase PI3K/AKT/mTOR inhibition with IBL-202 (PIM/PI3K) and IBL-301 (PIM/PI3K/mTOR) and that showed cell death of neuroblast cells and reduced tumor growth however in MYCN-amplified high risk neuroblastoma had PIM3 expression that shows resistance against this therapy [26].  Chromosomal alterations in neuroblastoma In neuroblastoma deletion of the 1q chromosome is common in most primary neuroblastoma tumors and it is characterized by loss of heterogeneity [50]. MYCN gene is found on chromosome 2 and gain of chromosome 2 can be a very very high risk because it increases MYCN gene expression, together with ALK gene [51]. With 11q gene alteration there is a study showing development of resistance against chemotherapy and 11q gene alteration and MYCN gene amplification is not linked at all [52]. Gain of the 17q chromosome is found out to be more occurrence than 11q chromosome alteration and 17q gain although identified in the smallest region but it’s associated with high-risk and relapse neuroblastoma [53].

Targeted therapy options

Therapy with low risk neuroblastoma non-MYCN amplified usually involves localized surgical resection with standard chemotherapy. This type of risk doesn’t metastasize and doesn’t spread after resection. For stage 1 patients with low risk is usually effective treatment [1]. With high risk neuroblastoma treatment is involved in several phases: induction phase, consolidation phase and the maintenance phase. In induction phase treatment is given with chemotherapy drugs like etoposide, vincristin, doxorubicin, cyclophosphamide and others. The consolidation phase includes chemotherapy along with radiotherapy and the maintenance phase is focused on retinoic acid treatment and immune activator drugs or cytokines that activate the immune system [54].

In conclusion, there was a lot of heterogeneity of MYCN genes and this type of heterogeneity often leads to high-risk neuroblastoma. There was also a lot of correlation of MYCN gene overexpression/amplification with different types of genes and with different molecular pathways. Although we mentioned many molecular mechanisms of genes and pathways and targeted therapy options that are related with neuroblastoma, there is still need for further research against targeted therapies and molecular mechanisms.