Exploring hot keywords, citation burst references, and trending topics aids in grasping the current research trends and frontiers within this domain (22). By analyzing through bibliometrics and visualization software, we summarize the hot topics in the academic field, objectively evaluate the forefront research directions in the field of NB omics. The current research focus includes the identification of potential biomarkers using omics in NB and the application of personalized medicine, as well as the challenges and future directions in NB omics.
Applications of omics in the identification of potential biomarkers and personalized medicine in NB
Our analysis of hot keywords, citation bursts, and trending topics reveals current research focuses on identifying biomarkers and personalized medicine in NB. The results highlight key genetic mutations and susceptibility loci, such as PHOX2B and ALK, which are central to familial and sporadic NB.
NB is categorized into familial NB and sporadic NB, with familial NB accounting for 2% of all NB cases (23). The first susceptibility gene mutation identified by NB is in PHOX2B, which contains two polyalanine repeat sequences. This gene encodes a paired-like homeobox transcription factor with functions that can promote cell cycle exit and neuronal differentiation (24, 25). The most common genetic mutation linked to familial NB occurs in the ALK receptor tyrosine kinase gene expressed in the sympathetic adrenal lineage during neural crest development (23, 26–28). This gene is implicated in modulating the equilibrium between proliferation and differentiation via diverse cellular pathways, including the mitogen-activated protein kinase (MAPK) and Ras-related protein 1 (RAP1) signaling pathways (29, 30). In addition, PHOX2B can directly regulate the expression of the ALK gene (31). The susceptibility single nucleotide polymorphisms (SNPs) associated with familial NB in genome-wide association studies (GWAS) include: BARD1, DUSP, DDX4-IL31RA, HACE1, HSD17B, LMO1, LIN28B, LINC00340, and LOC729177 (FLJ44180) (32–35).
In sporadic NB, PHOX2B mutations and GWAS susceptibility loci are infrequent. Activating mutations in ALK are found in about 6-10% of patients, while high-level ALK gene amplification is present in 3-4% of cases (23, 26, 36). The most common genetic mutation in sporadic NB involves the amplification of MYCN, expressed in the neural crest during development and plays a crucial role in regulating the proliferation, growth, differentiation, and survival of cells within the developing central nervous system. This amplification is observed in around 22% of patients and is linked to an unfavorable prognosis (37). Another common genetic mutation in sporadic NB is the ATRX mutation, and its relationship with the age of diagnosis is statistically significant. In NB patients harboring ATRX mutations, 17% manifest in children aged 18 months to 12 years with stage 4 disease, while 44% are observed in patients aged 12 and above (38).
Gene amplification in cancer and chromosomal aberrations may result in the dysregulation of messenger RNA, microRNA, and other non-coding RNAs, thereby disrupting cell apoptosis, differentiation, and immune surveillance (39, 40). The adverse prognosis in NB patients is specifically marked by deletions of 1p, 11q, and amplification of 17q. Additionally, deletions of 3p, 4p, 9p, and 14q, as well as amplifications of 1q, 2p, 7q, and 11p, also impact the prognosis (41, 42). The patient’s age, disease stage at the time of diagnosis, presence of MYCN amplification in NB cells, deletion of the 11q chromosome, histological characteristics, and ploidy have become crucial factors for stratifying risk groups in NB patients (43). High-risk NB patients have chromosomal rearrangements, and the frequency of these aberrations rises with the age at diagnosis, exerting a strong predictive impact on outcomes (44, 45).
Like genomics, proteomics research plays a crucial role in understanding the mechanisms of tumor formation in NB, discovering biomarkers, and elucidating pathways related to treatment response and drug resistance. Hsu’s study revealed a significant upregulation of GRP75, heat shock protein 2, protein disulfide isomerase A3 precursor, TCP1-containing chaperonin protein subunit 1β, and Eno1 protein during NB differentiation induction (46). GRP75, a member of the heat shock protein 70 family, is thought to function in tumor development control. Additionally, GRP78, another family member, together with GRP75, acts as a positive prognostic marker for NB in a separate study (47, 48). The mechanism involves the formation of the GRP75/78-RHAMM complex, which binds to hyaluronic acid-mediated motility receptor (RHAMM) associated with the progression and metastasis of cancer, and interacts with microtubules to stabilize them during the interphase, preventing microtubule depolymerization and promoting the progression of mitosis (49, 50).
Valérie employed surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to detect serum amyloid A (SAA) protein, revealing its close association with the prognosis of NB patients (51, 52). Targeted therapy for tumor-specific mutations can effectively and precisely eradicate cancer while sparing patients from the acute and chronic toxicities that may occur during chemotherapy and radiation therapy. However, in NB, traditional immunotherapy models are not applicable. Treatment approaches based on antibodies rather than T cells, especially those targeting cancer embryonic differentiation antigens, offer viable alternative strategies (21). Currently, research has found that the construction of T-cell vaccines targeting cytotoxic T lymphocytes (CTL) recognition, including cancer testis antigens (MAGE and NY-ESO-1), MYCN 110, and surviving in NB antigens, can stimulate T cell-mediated immunity to some extent (53–55).
GD2, a cancer embryonic differentiation antigen, is expressed not only during fetal development, but also in mature neurons, pain fibers, and skin cells (56). Treatment with anti-GD2 monoclonal antibodies (MAb) administered intravenously, particularly the anti-GD2 IgG antibodies (ch14.18 and murine 3F8), especially in combination with interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), and oral 13-cis-retinoic acid (CRA), can effectively alleviate symptoms in high-risk NB patients. The potential mediating mechanism is granulocyte-mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) and NK-ADCC (57, 58). Due to the depletion of white blood cells required for ADCC after induction chemotherapy or autologous stem cell transplantation, the combination of cytokines IL-2 and GM-CSF can be used with monoclonal antibodies (MAb). IL-2 can activate NK cells, natural killer T (NKT) cells, T cells, and regulatory T cells (T reg). Similarly, IL-15, like IL-2, can activate NK, NKT, and CD8+ T cells. However, in non-human primates studies, IL-15 does not lead to capillary leakage, activation-induced cell death, or an increase in T reg activation (59).
Proteomics research emphasizes the importance of biomarkers like GRP75 and GRP78 in NB. This is corroborated by Hsu’s research on these proteins and their roles in tumor development and prognosis. Our discussion on targeted therapies and antibody treatments aligns with current research trends and highlights advancements in personalized treatments for NB, such as the development of T-cell vaccines and anti-GD2 monoclonal antibodies. Zhang combined metabolomics and transcriptomics analyses, uncovering noteworthy distinctions in the cAMP, PI3K-Akt, and TNF signaling pathways between high-grade NB (HG-NB) and low-grade NB (LG-NB). HG-NB corresponds to stage 4 according to the International NB Staging System (INSS), while LG-NB includes stages 1, 2, and 3. Additionally, three biomarkers, MGST1, SERPINE1, and REBB3, were identified (60). The specific mechanism centers around cAMP as a second messenger, engaging in diverse cellular processes like growth, differentiation, and gene transcription. It influences four effector proteins, namely cAMP-activated exchange proteins, cyclic nucleotide-gated ion channels, myosin heavy chain proteins, and the cAMP-dependent PKA pathway (61, 62). The PI3K-Akt pathway exerts its influence on tumors through the stimulation of receptor tyrosine kinases and somatic mutations in pathway-specific components in somatic cells. Inhibiting this pathway can halt the progression of cancer (63). The dysregulation of PI3K-Akt in HG-NB has been linked to PTEN tumor suppressor gene dysfunction in several studies (64). In addition, a study found that activation of the PI3K-Akt signaling pathway can prompt cell death by suppressing autophagy (65). TNF plays a role in both tumor angiogenesis and cell death, promoting the progression and metastasis of tumors (66). Moreover, TNF can stimulate the expression and activation of downstream molecules, such as nuclear factor κB and p38 mitogen-activated protein kinase, thereby controlling diverse biological processes, including cell death (67).
The analysis conducted by Prasinou using fatty acid methyl ester (FAME) gas chromatography revealed that in apolipoprotein E (apoE), both apoE3 and apoE4 increased the levels of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA), as well as the omega-6/omega-3 ratio, in NBSK-N-SH cell membranes. They reduced total polyunsaturated fatty acids (PUFA), leading to decreased membrane stability indicators like PUFA balance, unsaturation index, and peroxidation index. Meanwhile, ApoE3 elevated stearic acid and dihomo-γ-linolenic acid (DGLA) levels, with apoE4 exhibiting the opposite impact. This study emphasizes the importance of membrane lipidomics in understanding the role of apolipoproteins in neurodegenerative diseases (68).
The integration of metabolomics and transcriptomics, as discussed by Zhang, emphasizes the importance of pathway analyses in distinguishing between high-grade and low-grade NB. This reflects our results on the differential signaling pathways and biomarkers associated with various NB grades. Radiomics, a swiftly advancing domain, concentrates on the extraction and analysis of numerous quantitative features from medical images. Utilizing advanced image processing algorithms, radiomics transforms medical images into rapidly exploitable data (69). These characteristics encompass information about tumor morphology, voxel texture, intensity, and spatial relationships within the tumor and its surrounding environment. In NB, various medical imaging modalities are currently employed, including computed tomography (CT), positron emission tomography-computed tomography (PET-CT), magnetic resonance imaging (MRI), and metaiodobenzylguanidine (MIBG) scintigraphy.
The International Neuroblastoma Pathology Classification (INPC) is an important classification system for NB. It categorizes NB into two subgroups, favorable histology (FH) and unfavorable histology (UFH), based on several risk factors linked to poor prognosis such as patient age, tumor histologic subtypes, differentiation grade, and mitosis-karyorrhexis index (MKI). This classification system provides a reference for different treatment stratifications (70). The pathological heterogeneity of NB results in inconsistent outcomes when multiple pathologists analyze the same patient. The heterogeneity within tumors results in diverse levels of differentiation across different tumor regions, diminishing the accuracy of the INPC. Assessing the MKI proves challenging and subjective, primarily due to the requirement for manually counting 5000 cells under a microscope (71). However, radiomics can automate and objectively extract quantitative features to reflect the heterogeneity of lesions, providing significant advantages in tumor staging, pathological subtypes, and prognosis prediction (72).
Studies indicate that radiogenomics has the potential to identify the pathological subtypes and genetic aberrations of NB (73). WU has developed a radiomics model based on CT images to predict MYCN amplification in pediatric NB. In the training group, the model achieved an Area Under the Curve (AUC) of 0.93, compared to 0.92 in the testing group (74). Wang employed a CT-based radiomics method for predicting the INPC of NB. The AUC values in the training and testing groups were 0.851 and 0.816, respectively. Decision curve analysis further validated the radiomics model’s outstanding performance across various high-risk thresholds (75). Qian created a radiomics model utilizing 18F-FDG PET/CT images for distinguishing INPC subgroups. The model demonstrated AUCs of 0.877 and 0.868 in the training and validation cohorts, respectively (76).
Challenges and future directions in NB omics
Due to the smaller number of pediatric patients compared to adults and significant differences in drug metabolism, acute toxicity, and late effects between children and adults, the translation and clinical research of pediatric cancer face a series of challenges compared to adult cancer. Additionally, most preclinical studies of pediatric cancers use murine cell lines or xenograft models, but for immunological analysis, mouse effector cells are not ideal human surrogates. To address this issue, it is recommended to use humanized mice implanted with human immune cells to better simulate the human immune system. The high-throughput capabilities of proteomic technologies provide a rich source for discovering potential biomarkers. In the future, NB proteomics can explore biomarkers through the study of protein fragment subtypes, glycoproteins, phosphorylated proteins, and autoantibodies (77). In future experiments using mouse models, it is possible to test the rational integration of antitumor monoclonal antibodies, effector cells, and cytokines for inducing tumor cell death. Additionally, the inclusion of small molecule drugs can be explored to prevent the escape of cell death (21).
The radiomic approach in NB holds the potential to enhance diagnostic accuracy, treatment planning, and prognosis prediction. However, realizing these goals faces several challenges. Firstly, the limited sample size of NB, due to its relative rarity, makes it difficult to obtain large sample cohorts with sufficient clinical and imaging data for radiomic analysis. Collaborative efforts, data sharing, and multi-institutional cooperation are essential means to overcome this limitation. Secondly, despite the quantitative nature of radiomics, transforming these features into biologically relevant information remains challenging. Lastly, the inherent heterogeneity in imaging protocols and data collection methods, stemming from multiple centers, may impact the reproducibility of research results. Implementing standardized imaging protocols, promoting the sharing of imaging datasets, and developing coordinated technologies are effective strategies to address this issue.
Another current issue is that most preclinical studies on childhood cancers, including NB, are short-term, utilize clinically irrelevant drug doses and regimens, fail to adequately consider combination chemotherapy, and lack appropriate benchmarks. Moreover, these studies often lack statistical design, proper randomization of animal groups for treatment plans, and mechanisms to keep the research team “blind” to the treatments administered to the study subjects. To address these issues, future efforts should involve building interdisciplinary teams comprising clinical researchers, laboratory scientists, pharmacologists, and biostatisticians.
The future direction of NB omics research should pivot towards interdisciplinary translational research teams that utilize validated preclinical models. These teams seek to pinpoint optimal combinations of molecularly targeted therapies, broad-spectrum chemotherapy, and immunotherapy to treat patients with unfavorable prognoses. Simultaneously, they aim to scale down treatment intensity for individuals with more favorable prognoses. The primary objective is to prioritize targeted therapies and minimize cytotoxic treatments, with the overarching goal of curing NB and mitigating treatment-related adverse effects in pediatric patients. To advance treatment strategies for NB, it is critical to delve deeper into the molecular mechanisms of NB tumorigenesis and progression, identifying key signaling pathways and molecular targets, and understanding the tumor microenvironment’s role in NB progression and metastasis. Developing novel therapeutic strategies, such as targeted therapies and immunotherapies, is essential to improve the prognosis for high-risk NB patients. Personalized medicine approaches that consider the genetic and molecular heterogeneity of NB tumors are also crucial. Addressing the challenges of tumor heterogeneity requires strategies to target diverse subpopulations of tumor cells within a single NB tumor, including combination therapies and single-cell omics approaches. Standardizing omics data processing and analytical methodologies is vital to ensure the reproducibility and reliability of research. This includes developing standardized protocols for sample collection, data processing, and analysis, as well as establishing large-scale, multi-center omics databases to validate findings and improve the reliability of NB omics research.