Role of Genetic and Epigenetic Alterations in Pathogenesis of Neuroblastoma

Introduction

Neuroblastoma is a sympathetic nervous system malignant tumor, which is derived from the neural crest cells and constitutes 7%–11% of the total number of childhood malignant tumors, taking fourth place in the structure of cancer morbidity after acute leukemia, central nervous system tumors, and malignant lymphomas. The incidence of neuroblastoma constitutes 0.85–1.1 cases per 100000 children under 15 years . The disease age distribution is heterogeneous; the frequency of tumor detection decreases with age, and 90% of patients are newborns and children up to 6 years. In children older than 14 years, neuroblastoma occurs rarely. Despite the comparatively low level of the disease, about 15% of pediatric cancer deaths are associated with neuroblastoma .

Unrestrained growth is the hallmark of the cancer cell. Carcinogenesis proceeds through the accumulation of genetic and epigenetic changes that allow cells to break free from the tight network of controls that regulate the homeostatic balance between cell proliferation and cell death . This property is caused by abnormal regulation of genes, which are involved in the control of cellular proliferation and cell death. Other disturbed cellular functions important for the evolution of cancer involve the control of DNA integrity, angiogenesis, and cellular senescence . It appears that the cause of these functional disturbances is mainly genetic rearrangements, although recently there is greater awareness of the possibility that stable epigenetic defects may contribute significantly to tumorigenesis, especially, in the case of childhood tumors of embryonic origin.

A characteristic feature of neuroblastoma is its clinical heterogeneity — from localized tumors to widespread forms and early hematogenous metastasizing. This high clinical heterogeneity reflects the complexity of genomic abnormalities characterized in neuroblastoma tumors . As a genetically complex cancer, neuroblastoma also displays significant genetic heterogeneity. That is why strikingly different outcomes are observed across tumor subtypes — from spontaneous regression without therapy to rapid progression and death due to the disease .

Individual tumors show great heterogeneity in their patterns of genetic alterations, epigenetic changes, and gene expression, even within homogenous histological groups . In addition, the fact that malignant phenotypes can be maintained solely by a tiny subpopulation of cells with stem cell properties , have proved that tumor heterogeneity is not merely a consequence of mutation acquisition, leading to a clonal expansion of mutated cells. Thus, it is now clear that there are multiple mechanisms by which cells can progress into malignancy, and that it is the concerted accumulation and functional cooperation between genetic and epigenetic changes, rather than their order of occurrence that drives carcinogenesis .

The role of MYCN gene amplification in neuroblastoma pathogenesis was first established in the early 1980s due to its association with high-risk tumors and low patient survival . Since then, several other genetic abnormalities were associated with neuroblastoma, including gains of whole chromosomes and a large number of large-scale chromosomal imbalances, such as loss of heterozygosity (LOH) at chromosome arms 1p, 3p, 14q, and 11q, unbalanced gain of 1q, 11p, and 17q and numerous mutations in key genes such as ALK, PHOX2B , and PTPRD .

Despite the extensive knowledge of the somatically acquired genomic rearrangements in neuroblastoma and their correlation with the clinical tumor phenotype, very little is known about the factors leading to these genetic events. Some changes in early embryogenesis or germ line are probably necessary for the development of neuroblastoma. A recent study has shown significant opportunities for epigenetic factors to promote carcinogenesis, especially in cases of childhood tumors with embryonic origin . The research on epigenetics has become increasingly visible because of the remarkable progress in our understanding of the critical role of epigenetic mechanisms in normal cellular processes and the abnormal events that lead to diseases, most notably, to cancer. Thus, genetic and epigenetic events represent two complementary mechanisms that are involved in every step of carcinogenesis, from responses to carcinogen exposures to progression into malignancy. However, we do not know what initiates this process in most cases, except in some inherited forms of cancer and some types of adult cancer in which environmental factors play a crucial role .

Epigenetics is defined as the study of heritable changes in the functioning of genes that occur without changes in the DNA sequence. Epigenetic modifications consist mainly of DNA methylation, histone modification, chromatin reorganization, and expression of non-coding RNA. Epigenetic modifications are well known as regulators of tissue-specific gene expression, genomic imprinting, and X-chromosome inactivation . In addition, the key role of epigenetic modifications during cellular differentiation, development, and organogenesis has been highlighted by the identification of many epigenetic biomarkers in human diseases, such as neuroblastic tumors .

The occurrence of many cancers is the result of the accumulation of genetic and epigenetic changes. While genetic alterations are nearly impossible to reverse, epigenetic changes can dynamically respond to signals from the physical, biological, and social environments . This characteristic confers the importance of epigenetic research in various cellular processes, particularly, in the regulation of gene expression. Although epidemiological data provide evidence that there is a direct interaction between epigenetic modifications and environmental influence on gene expression, the mechanism of epigenetic induced modulations of gene expression is still poorly understood .

Numerous studies have demonstrated that genomic and transcriptomic profiles can be predictive for clinical disease course, so that the combination of mRNA, miRNA, and comparative genomic hybridization are now being used to better define prognostic markers that could provide insight into the molecular basis of clinical heterogeneity in neuroblastoma . It is reflected in the International Neuroblastoma Risk Group (INRG) Staging System, which takes into account both clinical characteristics and tumor biology to identify clinical risk groups with statistically different survival rates. Independently prognostic baseline characteristics of this system included patient age, stage of disease, histology, grade of differentiation, DNA index, MYCN gene amplification, and presence of chromosome 11q copy number aberrations .

Genetic Alterations in Neuroblastoma Pathogenesis

In cancer cells, somatic mutations occur and accumulate at a significantly higher rate than in normal cells, a property referred to as “Mutator Phenotype.” This ability of cancer cells to accumulate mutations is critical for the development of cancer as well as for the rapid development of resistance to cytotoxic cancer treatments . The Mutator Phenotype can be caused by a number of mechanisms, such as defects in cell-cycle regulation, apoptosis, specific DNA repair pathways, or error-prone DNA polymerase, and it can have its source in inherited genetic defects that make the subjects prone to specific cancers.

Mutations in cancer cells cover a wide range of structural alterations in DNA, including changes in chromosomes copy numbers or chromosomal alterations encompassing millions of base-pairs such as translocations, deletions or amplifications, as well as smaller changes in nucleotide sequences such as point mutations affecting a single nucleotide at a critical position of a cancer-related gene . These different kinds of alterations often coexist within a single tumor.

While the origins of neuroblastoma tumorigenesis arise from the disrupted development of neural crest precursors, after the DNA and RNA sequencing of over 1000 cases, no single genetic or epigenetic mutation has been found to account for all cases of neuroblastoma . Likewise, structural genomic changes have not been linked to neuroblastoma tumorigenesis. For example, 1p deletion, MYCN amplification, or gain of 17q may identify subtypes of neuroblastoma and impact survival , yet there is no common neuroblastoma-specific genomic alteration, LOH, or genetic translocation uniformly ascribed to all high-risk neuroblastoma tumors. Thus, this extensive molecular heterogeneity supports the concept that neuroblastoma represents a spectrum of diseases. Clinically, this presents a challenge as tumors that are phenotypically and morphologically very similar can have highly disparate responses to treatment. Consequently, extensive efforts have focused on characterizing the transcriptomes and oncogenic pathways, which are active in the most aggressive and fatal subtypes . In addition to elucidating the genetic and epigenetic origins of neuroblastoma, these efforts are motivated by the potential to yield actionable therapeutic targets for this highly fatal cancer. Over the last three decades, many chromosomal and molecular abnormalities have been identified in patients with neuroblastoma. These biological markers have been evaluated to determine their value in assigning prognosis, and some of these have been incorporated into the strategies used for risk assignment.