Advances in liquid biopsy in neuroblastoma

Abstract

Even with intensive treatment of high-risk neuroblastoma (NB) patients, half of high-risk NB patients still relapse. New therapies targeting the biological characteristics of NB have important clinical value for the personalized treatment of NB. However, the current biological markers for NB are mainly analyzed by tissue biopsy. In recent years, circulating biomarkers of NB based on liquid biopsy have attracted more and more attention. This review summarizes the analytes and methods for liquid biopsy of NB. We focus on the application of liquid biopsy in the diagnosis, prognosis assessment, and monitoring of NB. Finally, we discuss the prospects and challenges of liquid biopsy in NB.

Graphical abstract

1. Introduction

Neuroblastoma (NB) is an embryonic neoplasm of the sympathetic nervous system [1]. Primary NB arises from incomplete precursors of neural crest tissue [2], [3], [4]. It usually forms in the adrenal medulla [4], [5], [6], [7]. Abnormalities in genome, epigenome, transcriptome, and protein expression levels were identified in NB [8], [9], [10], [11]. These abnormalities provide a basis for studying the genesis and development mechanism of neuroblasts. However, the specific mechanism of NB remains unclear. NB often occurs in infancy [12]. It is the second most common extracranial malignancy in children [12]. There were 10.2 cases of NB per million children under 15 years of age [3]. NB presents clinical heterogeneity due to biological heterogeneity [3,[13], [14], [15]]. NB is assessed for different risks based on different biological and clinical features [3]. The main categories are low-risk, medium-risk, and high-risk NB. Patients with low-risk NB can be treated with surgery, and some regress spontaneously [16]. The survival rate for low-risk NB patients is over 98% [3]. Moderate-risk NB patients are generally treated with mild chemotherapy or surgery, and the prognosis is good [3,16]. Although high-risk NB patients were treated with intensive chemotherapy, surgery, radiotherapy, and immunotherapy, the prognosis was still poor [3,[17], [18], [19], [20], [21], [22]]. There is no denying that the survival rate for high-risk NB patients has improved over the past 20 years, but it is still 40-50% [23], [24], [25], [26], [27], [28]. About half of high-risk NB patients relapse in the first 2 years after treatment [29,30]. Intensive treatments also have a negative impact on the quality of life in patients with NB [31]. The era of personalized treatment for NB patients is coming. The best treatment should be tailored to the biological and clinical characteristics of patients with NB.

At present, molecular targeted therapy for NB is a new effective treatment method. It focuses on genomic aberrations and signaling pathways [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42]. As we all know, tumor biopsy remains the gold standard for diagnosing primary or metastatic tumors, identifying tumor biology, and making treatment decisions. However, invasive biopsies are difficult to obtain in children with NB [43]. Due to the large heterogeneity of NB, the biopsy may have some limitations in identifying the whole NB [44]. Many medical facilities use meto-iodobenzylguanidine (MIBG) scans, Fluorodeoxyglucose positron emission tomography scans (FDG-PET), standard computed tomography (CT) scans, and magnetic resonance imaging (MRI) to assess the disease stage, treatment outcome, and prognosis of patients with NB [45]. However, these machines are not widely available. Therefore, non-invasive liquid biopsy has become a focus of attention.

“Liquid biopsy” is the detection of human information through body fluids which can be blood [46], marrow [47], spinal fluid [47], urine [48], etc. A large number of studies have shown that liquid biopsy plays an important role in the diagnosis, treatment, and prognosis of solid tumors [49], [50], [51], [52]. In recent years, many studies have carried out important research on NB based on blood and bone marrow [53,54]. In blood and bone marrow, we can analyze tumors by the dissemination, necrosis, and secretion of tumor cells [55,56]. For example, the importance of substances such as circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), and exosomes present in the blood in NB is being explored [57,58]. The presence of disseminated tumor cells in the bone marrow tends to show worse outcomes [59]. Blood-based liquid biopsy can reflect the heterogeneity of NB to a certain extent [60]. Liquid biopsy blood reflects only a fraction of tumor heterogeneity, but it has the advantage of being less invasive than tissue biopsy. Because NB is often difficult to obtain tissue samples, liquid biopsy techniques are an indispensable research tool for NB [61]. Blood-based assays of tumor cells, genetic material, and cellular secretions can analyze genomic, transcriptomic, proteomic, and epigenetic changes in patients at different disease stages [62]. This has important clinical value for the diagnosis, treatment, and prognosis of NB and other cancers [47,58]. Liquid biopsies have been used to detect biological changes in ALK inhibitor therapy to study resistance mechanisms [63]. Circulating tumor DNA has been shown to predict solid immunotherapy response in locally advanced non-small cell lung cancer [64]. ctDNA has been used in clinical trials to identify patients with HER2-mutant metastatic breast cancer [65]. Genomic and epigenomic alterations in ctDNA proved to be diagnostic markers for neuroendocrine prostate cancer [66]. Interestingly, immune cells in the blood have emerged as valuable tumor biomarkers [67]. One study showed that the activation of peripheral blood granulocytes is closely related to the prognosis of NB patients after immunotherapy [68]. The golden age of liquid biopsy for NB immunology is coming.

This review first describes analytes and techniques for peripheral blood biopsy of NB. We focus on describing the biological significance of liquid biopsy analytes in genomics, transcriptomics, proteomics, epigenetics, and immunology, and we summarize the important clinical implications of liquid biopsy in NB. Finally, we discuss the promise and challenges of liquid biopsy in NB.

2. Circulating analytes and techniques for liquid biopsy of NB

At present, the liquid biopsy of NB is mainly to analyze analytes in blood. These analytes mainly include CTCs (Circulating tumor cells), Exosomes, nucleosomes, cfDNA (circulating cell-free DNA), ctDNA (circulating tumor DNA), and cfRNA (circulating cell-free RNA) (Fig. 1). Several techniques have been developed for the detection and evaluation of liquid biopsy analytes. We describe some of the techniques that have been used or may in principle be used for the detection and evaluation of liquid biopsies in NB (Fig. 2 and Table 1). Circulating tumor cells and exosomes are purified and analyzed by isolation and enrichment techniques, followed by the analysis of DNA, RNA, and protein content. Nucleosomes were assessed by analysis of histone modification and nucleosome localization. Circulating DNA is analyzed by genomic and epigenetic tests. Circulating RNA was analyzed by reverse transcription gene sequencing.

Fig. 1.

Liquid biopsy analytes of peripheral blood of NB. Liquid biopsies in blood have been shown to contain single tumor cells (CTC), free nucleic acids (cfDNA and cfRNA), exosomes containing nucleic acids and proteins, and free circulating nucleosomes.

Fig. 2.

NB's liquid biopsy analyte detection technology. Techniques have been developed for the detection and evaluation of liquid biopsy analytes. (a-b) Circulating tumor cells (a) and exosomes (b) are purified and analyzed by isolation and enrichment techniques, followed by the analysis of DNA, RNAs, and protein content. (c) Nucleosomes were assessed by analysis of histone modification and nucleosome localization. (d) Circulating DNA is analyzed by genomic and epigenetic tests. (e) Circulating RNA was analyzed by reverse transcription gene sequencing.

Table 1.

Summary of liquid biopsy technologies for NB. 

Analyte	Method	Specific methods used	References
CTCs	Separation and enrichment	Cell isolation methods include immunoaffinity or size-selection in combination with flow cytometry, such as Imagestream Imaging flow cytometry	[53]
	Detection and assessment of contents (DNA, RNA, proteins)	ELISPOT detects proteins based on antibody capture	[81]
		Smart-SEQ analyzes the entire transcriptome of a single cell	[82]
Exosomes	Separation	Continuous differential centrifugation, density gradient ultrafiltration, antibody-based affinity purification, or ultrafiltration	[43,85]
		NPLEX can be used for high throughput quantitative analysis of unlabeled exosomes	[90]
	Detection and assessment of contents (DNA, RNA, proteins)	A 2100 bioanalyzer analyzes RNAs	[89]
Nucleosomes	Histone methylation	ELISA (enzyme-linked immunosorbent assay)	[95]
	Nucleosome footprinting	Deep sequencing NGS	[97]
Circulating DNA (cfDNA, CTC DNA, exosome DNA)	Genomic testing	Targeted PCR (qPCR, ddPCR, BEAMing)	[113], [114], [115]
		WES,WGS	[120], [121], [122], [123]
	Epigenetic detection	5mC detection (MSRE-PCR, MS-PCR, MeDIP-seq, T-WGBS)	[127], [128], [129], [130]
		methylation-specific PCR	[131]
Circulating RNA (cfRNA, CTC RNA, exosomal RNA)	transcriptomics	RT-PCR, RT-dPCR	[143]
		RNA-seq	[146]

2.1. CTCs

CTCs (circulating tumor cells) were reported by Ashworth et al. in 1869 [69]. They are defined as cells that circulate freely in the bloodstream and have a genetic similarity to their tumor origin (Fig. 1). One study showed that there were almost no CTCs in healthy subjects or patients with non-malignant diseases [70]. However, they are widespread in patients with metastatic cancer [70]. This suggests that CTCs may be the origin of metastatic disease. Patients with NB are often found to have CTCs in the blood during diagnosis and treatment [54,71]. Many studies have shown that CTCs can be used as circulating biomarkers for NB prognosis [54,72]. Therefore, CTCs are attractive analytes for liquid biopsy of NB [43]. CTCs are less abundant in the bloodstream. There are between 5 and 1281 CTCs per milliliter of blood [73]. CTCs are difficult to identify and capture, and many CTCs isolated by complex techniques have very low purity and low quantity [73,74]. Some emerging technologies are available to improve the efficiency of the separation and enrichment of circulating tumor cells, which provides good conditions for the counting and content analysis of CTCs (mainly DNA and RNA analysis) [43,73].

Isolation techniques based on tumor-specific markers have always been the focus of development. Separation of NB tumor cells in blood is also a prerequisite for the analysis of CTCs. After separation and enrichment of CTCs, effective detection can be carried out to further analyze the number of CTCSs and the biological information contained in their DNA and proteins (Fig. 2a). The CellSearch system (Silicon Biosystems) mainly uses the tumor cell marker epithelial cell adhesion molecule (EPCAM) to isolate and enrich CTCs [75,76]. It can separate enriched and count EPCAM-positive CTCs by immunomagnetic and immunofluorescent labeling [75]. CellSearch system has been approved by FDA to diagnose metastatic breast, prostate, and colorectal cancer [43,77]. However, NB lacks universal and specific tumor markers [45]. One study examined CTCs in the peripheral blood of patients with NB using an EPCAM-independent method [45]. This study used Cytelligen CTCs Enrichment Kit for CTCs enrichment. Then CD45 and DAPI immunostaining and chromosome 8 probe centromeric in situ hybridization (FISH) were performed to identify CTCs. Abnormalities of the CTC chromosome were identified by FISH analysis. More than 2.2% of cells with tri-signaled nuclei were considered to have abnormal trisomy 8 clones. It is exciting that systems have been developed to capture and enrich EPCAM-negative CTCs using physical principles, such as CellSieve™ (Creatv MicroTech) and ClearCell® FX (Genomax Technologies) [78,79]. At present, Imagestream Imaging flow cytometry (ISx) has been used to detect GD₂⁺/CD45⁻ CTC in the blood of patients with NB [53]. Swathi Merugu et al. first performed immunofluorescent antibody staining for GD2-percp, NCAM-PE, and CD45-PE-CY 7. The nuclei were then stained with DAPI. Finally, CTCs were detected based on GD₂ expression and CD45 expression [53]. After effectively separating the cells, further analysis of DNA and protein content can be carried out. Batch CTC analysis is very limited, it requires high yield and high purity samples [80]. Therefore, the detection of CTC content by the single-cell method is a research hotspot in recent years [80]. Currently, ELISPOT can be used to detect proteins secreted by circulating tumor cells [81]. It uses antibodies coated on cell membranes to capture proteins. Then a fluorescent dye-labeled secondary antibody was subsequently detected. The mRNA-SEQ protocol (Smart-SEQ) has been developed for the analysis of a single-cell whole transcriptome. Smart-seq has been applied to melanoma circulating tumor cells, and a new candidate biomarker of melanoma circulating tumor cells has been identified [82]. With the maturity of liquid technology, the detection of CTC in NB has great development potential and important application value in the future.

2.2. Exosomes

Exosomes are small extracellular vesicles secreted by cells (Fig. 1). They are lipid bilayer inclusions containing DNA, RNA, and proteins. There are MHC proteins in the membrane surface molecules of exosomes [43,83]. In particular, exosomes can be secreted by cancer cells and have become important substances in studying the mechanism of tumor progression in recent years [84]. Exosomes play an important role in mediating intercellular communication. For example, exosomes can facilitate the communication of signal RNAs, DNA, and proteins between cells [84,85]. Signal RNAs include long non-coding RNAs (lncRNA) and micro RNAs (miRNA) [43]. In other words, exosomes can mediate communication between tumor cells or between tumor cells and normal cells. Exosomes that are secreted by cancer cells can mediate tumor growth through such communication. However, some normal cells produce exosomes that mediate antitumor responses and suppress tumors [56]. Exosomes are often observed in the blood, and they are also present in urine, saliva, and cerebrospinal fluid [85], [86], [87]. In the early stage, the number or size of exosomes was mainly detected [88]. But measuring the number or size of exosomes alone is not enough to study tumors. At present, people mainly analyze the content of RNAs, DNA, and proteins of exosomes [43]. Exosomes were found in the plasma of patients with NB [89]. Intriguingly, the significantly upregulated expression of hsa-miR199a-3p in exosomes was found to correlate with the severity of NB patients [89].

Methods for exosome separation include continuous differential centrifugation, density gradient ultrafiltration, antibody-based affinity purification or ultrafiltration, and size exclusion chromatography [43,85]. Differential centrifugation can't separate exosomes by particle size completely due to the density difference of objects [85]. Density-based ultrafiltration makes up for this shortcoming. Ultrafiltration can separate exosomes more effectively. Because ultrafast centrifugation can not only separate the extracapsular protein complex and lipoprotein but also can separate the extracapsular pollutants through high-speed rotating precipitation [85]. However, ultrafiltration takes a lot of time and is not suitable for small clinical samples [85]. Immunoaffinity purification is based on capturing antibodies such as CD63, CD9, and CD81 to capture exosomes with these surface characteristics [85]. The traditional separation methods described above usually require extensive sample purification or biomarkers. nano-plasmonic exosome (nPLEX) analysis is a high throughput method for quantitative analysis of label-free exosomes [90]. NPLEX is used to separate exosomes through the principle of transmission surface plasmon resonance of periodic nanopore array. With the increasingly advanced technology for capturing and isolating exosomes, people mainly focus on the biological information contained in exosomes, such as the RNA and protein content contained in exosomes. A study isolated and purified plasma exosomes from NB patients using an exoRNeasy serum/plasma MIDI kit [89]. The exosomal RNAs were extracted by Qiazol and RNeasy MinElute Spin columns. This experiment then used a 2100 bioanalyzer to determine the content and quality of exosomal RNAs. Marta et al. [91] separated and purified the plasma exosomes of NB through the principle of continuous centrifugation, and the purified exosomes were characterized and verified according to the relevant biological markers of exosomes secreted by NB cells [92]. The content of exosomes is of concern (Fig. 2b). Marta et al. isolated RNA from exosomes for detection of miRNA expression using the Exosomal RNA Purification Mini Kit (serum format) according to the manufacturer's protocol [91]. In conclusion, the detection technology of circulating exosomes of NB is expected to become an important means to study NB.

2.3. Nucleosomes

We also see nuclear components in blood, such as circulating nucleosomes and histones (Fig. 1). Each nucleosome consists of 146bp DNA wrapped around 1.75 loops of the histone octamer. Histone modification plays an important role in epigenetics. For example, histone modification is an important way to regulate chromatin accessibility to gene expression [93]. Nucleosome localization is also involved in epigenetic gene expression [93]. Methylation can specifically activate or inhibit the transcriptional activity of genes [93]. There are few studies on circulating nucleosomes and histones in NB. Studies have shown that cell death causes chromatin to fragment [94]. DNA, Nucleosomes, and histones are shed into the bloodstream by fragmentation of chromatin. There are few studies on circulating nucleosomes and histones in NB. DNA and histone modifications in circulating nucleosomes may play a role in NB. Circulating nucleosomes and histones in NB remain to be studied.

Tumors have specific nucleosome positioning and histone modifications. Detection of nucleosome positioning and histone modifications in liquid biopsies is an important technique (Fig. 2c). Circulating nucleosomes are mainly carried by circulating cell-free DNA (cfDNA), but detection of cfDNA is not required to detect nucleosome and histone modifications. Circulating nucleosomes can be quantitatively and qualitatively detected using ELISA (enzyme-linked immunosorbent assay) [95]. Circulating nucleosomes have been shown to be potential cancer biomarkers [95]. Quantitative monitoring of circulating nucleosome levels is a means of observing tumor response [95]. Ugur Gezer et al. used EpiQuik Global Tri-Methyl Histone Quantification Kits (Epigentek, Farmingdale, NY, USA) to directly capture and analyze H3K9me3, H3K27me3, and H4K20me3-modified histones in plasma [96]. The localization of circulating nucleosomes is dynamic, and the broader nucleosome footprint is an emerging technique for studying circulating nucleosomes and cfDNA [97]. A study has shown that nucleosome occupancy mapping on circulating cell-free DNA is associated with gene expression and cancer type [98]. Deep sequencing of circulating cell-free DNA has been performed and nucleosome occupation maps of the whole genome have been generated [97].

2.4. cfDNA and ctDNA

Cancer patients' DNA, mRNA, and microRNA (miRNA) are released into the blood [99] (Fig. 1). The free DNA released into the blood is circulating cell-free DNA (cfDNA). The cfDNA has been shown to be free double-stranded DNA fragments carrying nucleosomes [97]. Apoptosis or necrosis of cancer cells releases cell-free DNA into the bloodstream [100]. DNA from tumor cells enters the circulatory system called circulating tumor DNA (ctDNA). Detection of circulating tumor cell DNA (ctDNA) can help assess tumor burden. Also, dying cells in the surrounding tumor microenvironment release DNA into the bloodstream [100]. Detection of cfDNA is of great significance for the study of tumors (Fig. 2d). DNA from the fetus has been found in the mother's blood, which is exciting news for noninvasive prenatal diagnosis [101]. From 0 to 1000ng of cfDNA and even more than 1000ng of cfDNA can be isolated per ml of plasma from cancer patients [99]. There is much less cfDNA in the plasma of healthy individuals, and 0-100 ng cfDNA can be extracted per milliliter of them [102]. Tumors are often heterogeneous, with cells in different regions having different mutation profiles [103]. For example, the biopsy might miss important mutations [104]. However, cfDNA can capture mutations at these different sites, making up for important tumor-related mutations that biopsies miss [105]. One study found a significant increase in cfDNA in patients with newly diagnosed NB compared with patients with stable NB [106]. Many of the high levels of cfDNA found in patients with NB at diagnosis are circulating tumor cell DNA (ctDNA), which is derived from the tumor [107]. Circulating tumor cell DNA (ctDNA) contains many important genomic alterations, such as MYCN amplification, ALK mutations, DNA methylation, deletions of 1p, 3p, and 11q, and increases of 1q, 2p, and 17q [60,108,109]. These mutations have important clinical implications for the diagnosis, risk stratification, treatment response monitoring and prognosis of patients with NB [44,110].

With the development of DNA extraction and isolation technology, a variety of commercial kits for the extraction of cfDNA and ctDNA have emerged for different experimental purposes. Because serum contains a large amount of leukocyte-lysed DNA, plasma is generally used as the source of cfDNA and ctDNA extraction [58]. Column-based extraction methods cause too much loss, and magnetic bead-based methods have been developed to extract cfDNA and ctDNA based on average ctDNA length, density, and particle conductivity [111]. Cancer-related mutations, copy number variations (CNV), or single nucleotide polymorphisms (SNPs) in circulating cell-free DNA (cfDNA) can be assessed using PCR-based targeting or genome-wide next-generation sequencing (NGS) methods. DNA in circulating tumor cells and exosomes can also be analyzed using these techniques. next generation sequencing (NGS) is high-throughput Sequencing, which can greatly reduce the cost and time of sequencing [112]. Cobas ® EGFR Mutation Test V2 is a real-time PCR test (qPCR) approved by the FDA for liquid biopsy in patients with NSCLC (non-small cell lung cancer). It can detect mutations in EGFR in patients with NSCLC [113]. The qPCR is usually limited by the low plasma ctDNA content of certain cancer patients. The Digital PCR platform can make up for this shortcoming, it has the characteristics of high sensitivity analysis. For example, microfluidic droplet digital PCR (ddPCR) and BEAMing (beads, emulsions, amplification, and magnetism) are more sensitive to the detection of specific mutations and deletions [114,115]. A recent preclinical trial demonstrated that droplet digital PCR (ddPCR) can be used to accurately determine the MYCN and ALK copy number status of cfDNA in plasma of patients with NB [116]. Microsatellite analysis based on PCR technology has also been used for liquid biopsies of circulating DNA [117]. Microsatellites are short DNA sequences consisting of nucleotide sequences of 1∼6 bases in length, which are ideal genetic markers [118]. One study demonstrated the use of microsatellite analysis to detect 11q loss of ctDNA in serum in NB [117]. The qPCR and ddPCR mainly analyze a single mutation region, and next-generation sequencing (NGS) can analyze multiple mutations. Forshew et al. developed the TAM-SEQ technique to analyze six genes to detect low-frequency mutations in cell-free DNA [119]. The InVision™ liquid biopsy platform was recently developed, which utilizes enhanced TAM-SEQ ™ technology based on a next-generation sequencing approach to identify low-frequency clinically relevant somatic changes in ctDNA [113]. This study detected 90 percent of the mutations in samples with low amounts of incoming DNA. There are whole exon sequencing (WES) and whole genome sequencing (WGS) based on NGS. The coding region of exons accounts for about 1% of the genome [120]. Total exon sequencing (WES) provides a method to study exons of circulating DNA by detecting the coding regions of exons of all genes [120]. WES mostly tests for known mutations and CNV in liquid biopsies. A recent study conducted WES analysis in patients with NB [121]. This experiment found that tumor tissue and plasma cfDNA had a highly consistent 17Q gain status. However, the coverage depth of WES is usually lower than that of targeted PCR sequencing and its ability to detect allele mutations is limited when the frequency is less than 5% [122]. Whole genome sequencing (WGS) not only sequenced coding regions contained in exons but also analyzed non-coding regions [123]. This allows a wider range of gene mutations, CNVS, SNPs, and larger structural changes to be assessed. WGS sequencing coverage is generally lower than WES. With the development of NGS, the cost of sequencing has been greatly reduced. WES and WGS remain the focus of research techniques for liquid biopsies of circulating DNA. In conclusion, it is necessary to develop more sensitive and specific DNA testing technology for liquid biopsy in the future.

Genomic detection methods have always been limited by low-frequency mutations. Epigenetic modification is common in DNA, and different cells have specific epigenetic markers [124]. Epigenetic analysis of circulating DNA can reflect metabolic and immune responses of cancer, and epigenetic characteristics have important clinical value in cancer diagnosis, prognosis, and recurrence detection [125]. DNA modification is often cytosine methylation (5mC). It happens all over CpG Island. Cytosine methylation (5mC) is involved in transcriptional inhibition [43]. Abnormally increased expression of focal 5mC is associated with cancer occurrence, progression, and invasion [126]. DNA methylation in specific regions of single or multiple fragments can be detected using Methylation sensitive restriction enzyme digestion PCR (MSRE-PCR) or methylation-specific-PCR (MS-PCR) [127]. Genome-wide DNA methylation is a technique for detecting DNA methylation status in single or multiple regions of the genome. Methyl-seq is a genome-wide DNA methylation detection technique developed based on MSRE and NGS [128]. It can detect more than 250,000 methyl-sensitive restriction enzyme cutting sites. Methylated DNA immunoprecipitation sequencing (MeDIP-seq) based on MeDIP and DNA sequencing dream detects genome-wide methylation using lower DNA inputs [129]. Traditional whole-genome bisulfite sequencing requires many DNA samples to detect genome-wide DNA methylation status [130]. Marker-based WGBS (T-WGBS) greatly reduces the amount of input DNA required [130]. In a study investigating the gene methylation status of cfDNA, DNA was first extracted from pretreated serum samples by the QIAmp DNA Blood Mini Kit (Qiagen, Hilden, Germany), followed by methylation-specific PCR to detect NB cfDNA methylation status in patient serum [131]. In this study, the methylation status of RASSF1A was associated with prognosis. Similarly, Shigeki et al. first extracted and isolated cfDNA then treated the DNA with sodium bisulfite recommended by the reagent manufacturer, and added methylation-specific primers for PCR detection [109]. They found that abnormal hypermethylation of the DCR2 promoter is associated with a poor prognosis of NB. In conclusion, epigenetic detection is a promising field for liquid biopsy, and epigenetic analysis of circulating DNA in NB has potentially important clinical value.

2.5. cfRNA

Cell-free long non-coding RNA (lncRNA), microRNAs (miRNAs), and messenger RNA (mRNA) are also shed by dying tumor cells or other dying cells [43]. In 1999, cell-free tyrosinase mRNA was found in the serum of patients with malignant melanoma and was found to be able to be amplified [132]. This discovery provides a new way to explore the application of circulating cell-free mRNA in cancer. The length of lncRNA was over 200 bp. The lncRNA is involved in the regulation of many intracellular processes, especially epigenetic regulation [133]. Many studies have detected lncRNA in the plasma of cancer patients [134,135]. The lncRNA is also present in exosomes. The lncRNA accounted for 3.36% of total exosomal RNA [136]. The lncRNA expression has been proved to be associated with the survival prognosis of patients with non-small cell lung cancer and metastasis of patients with breast cancer [137,138]. With the continuous development of detection methods of circulating non-coding RNA, the study of lncRNA in tumor patients' serum will be further deepened, and circulating non-coding RNA will play an important role in patient diagnosis and prognosis. MicroRNAs (miRNAs) are non-coding RNAs about 20-25 nucleotides in size that are involved in the post-transcriptional regulation of gene expression [139]. The miRNA expression in human cancers is often disrupted by the amplification or deletion of miRNA genes [139]. Changes in miRNA expression can cause biological changes in tumor development. Altered miRNA expression is associated with biological characteristics of tumor development, such as maintenance of escape growth inhibitors, resistance to cell death, activation of invasion and metastasis, and induction of angiogenesis [139]. Many studies have found that serum miRNA levels are associated with prognosis and treatment response in cancer patients [140]. High expression of miRNA-9-3p has been found in the serum of patients with MYCN-amplified NB [141]. High miRNA-9 expression is associated with metastatic disease [142].

Since RNA can be synthesized by reverse transcription of complementary DNA (cDNA), targeted PCR or more broadly sequencing can analyze RNA by detecting cDNA (Fig. 2e). Specific cancer-associated RNA alterations can be detected by quantitative reverse transcriptase PCR (RT-PCR) [143]. In a trial of children with stage 4 NB, quantitative real-time reverse transcriptase PCR (RT-qPCR) was used to detect mRNA in pre-isolated peripheral blood and bone marrow aspirate [144]. This experiment found high levels of TH and PHOX2B mRNA. In the case of low input samples, droplet digital PCR can be applied to RT-PCR to improve detection efficiency [145]. Sequencing methods for evaluating cDNA of circulating RNA include transcription microarrays and whole-transcriptome high-throughput sequencing. RNA-seq (RNA sequencing) represents a recently developed deep sequencing technique for circulating RNA [146]. The mRNA-Seq (Smart-Seq protocol is a powerful technique for detecting mRNA levels in single cells [82]. This brings good news for the detection of rare cells. Smart-seq has been used to detect mRNA from circulating melanoma cells and identify different gene expression patterns [82]. In conclusion, some techniques have been used to detect and analyze the expression status of relevant circulating RNAs in NBs with promising findings. Circulating analytes and analytical techniques require more preclinical and clinical validation.

3. Biological alterations found in the liquid biopsy of NB

We provide a brief overview of the definitions, sources, biological significance, and detection techniques of important liquid biopsy analytes above. Below we highlight the genomic, transcriptomic, and epigenetic alterations found in NB liquid biopsies. We focus on the reliability and specificity of biological alterations found by liquid biopsy. For example, we explored how consistent the mutations detected by liquid biopsy analytes were with those detected by tissue biopsy or other methods. In particular, the mutations found in tissue biopsy were specifically associated with NB. In addition, we also focus on the important biological roles of some new biological changes discovered by liquid biopsies in tumors., which may explain part of the tumor relevance of specific changes. In conclusion, the biological alterations identified by liquid biopsy may drive basic research on the mechanism of action of liquid biopsy in NB and even other tumors.

3.1. Genomic alterations

Analysis of tumor genome alterations is generally accomplished by assaying DNA in formalin-fixed paraffin-embedded (FFPE) tumor tissue samples [147,148]. However, this analytical procedure needs to be improved in terms of the quality of extracted DNA and the uniformity of detection methods [149]. Importantly, owing to tumor heterogeneity and cloning molecular evolution, the biological information contained in local biopsy tissue DNA cannot completely reflect tumor-related genome changes [10,150,151]. However, liquid biopsy has emerged as a promising modality for analyzing tumor-associated genomic alterations. In contrast, the analysis of tumor-related genomic alterations by liquid biopsy has become a very promising modality. Although liquid biopsy detection of genomic mutations is suitable for advanced tumors or childhood tumors that cannot be biopsied, whether the genomic mutations contained in liquid biopsy analytes can reflect tumor-related mutations is a question worthy of consideration [152], [153], [154]. Several of the liquid biopsy analytes described above contain genomic information, such as CTCs, exosomes, cfDNA, and ctDNA. In particular, cfDNA and ctDNA have become the most commonly used analytes for liquid biopsy analysis of genomic information [155].

Mathieu Chicard et al. analyzed and compared genomic alterations in primary tumors and cfDNA at diagnosis and at other different time points thereafter [121]. At diagnosis, their analysis by the Sequenza tool [156] found that 15%–98% of tumor cells in primary tumor samples (73% on average) and 3%–99% of blood cfDNA samples were ctDNA (60% on average). On average, 19 single nucleotide variants (SNVs) were shared per primary NB and cfDNA sample, including variants in the ARHGAP gene, the gene ALK, the MAPK pathway gene, the MLL gene, and the NOTCH gene (Table 2). ALK mutation is the most common NB mutation and can promote NB proliferation through multiple signaling pathways (Table 2) [63]. Activation of the MAPK pathway is associated with malignancy in NB (Table 2) [157]. Interestingly, an average of 22 SNVs per case were found only in cfDNA, including ATXNL3, RUNX3, GATA6, ARHGAP6, MLL4, NKTR, ERG, RASA1, DICER1, ARHGAP18, NOTCH2, SHH and other genes of the MAPK pathway. RUNX3 was previously shown to be an inhibitor of NB and has an important role in NB (Table 2) [158]. NB patients with reduced expression of DICER1 tend to have a worse prognosis (Table 2) [159]. NOTCH2 is involved in neuroblastoma-promoting signaling (Table 2) [160]. At diagnosis, they also found that most copy number variations (CNVs) were present in primary NB. Of these, the recurrent alterations seen only in cfDNA are 1p deletions and 17q gains. A recent study showed that cfDNA detected undetectable MYCN-amplified and ALK-altered cell clones in tumor tissue samples in a cohort study (Table 2) [55]. Flora Cimmino et al. have demonstrated some important biological mutations associated with NB, such as ALK, FGFR1, KMT2C, CREBBP, NOTCH1/2, ARID1A/B, CARD11, and FAT4 mutations, can be reliably detected by detecting ctDNA of NB patients [62]. ALK R1275 mutations are common in neuroblastoma [161]. The ALK R1275G mutation activates the EGFR pathway in NSCLC [162], and activation of the EGFR pathway is often associated with malignant tumor progression [163]. FGFR1 (fibroblast growth factor receptor 1) is involved in cancer epithelial-mesenchymal transition (EMT) and promotes cancer metastasis [164]. FAT4 was shown to inhibit cell invasion, migration, and proliferation by promoting autophagy by regulating the activity of PI3K [165]. In the future, it is expected to further study the specific mechanism of action of these mutations in NB.

Table 2.

Biological alterations found in NB liquid biopsy. 

Analyte	Type	Biological change	Observed	Correlation	References
cfDNA	SNV	ALK	1	Promote NB growth	[63,121]
		MAPK pathway genes	1	Be associated with NB malignancy	[121,157]
		RUNX3	2	Suppress NB growth	[121,158]
		DICER1	2	Be related to poor prognosis	[121,159]
		NOTCH2	2	Involved in the cancer-promoting pathway of NB	[121,160]
	CNV	MYCN amplification	2	Be related to poor prognosis	[55,121]
CTCs	SNV	ALK	/	Promote NB growth	[53]
	CNV	MYCN amplification	/	Be related to poor prognosis	[53,121]
cfRNA	Abnormal mRNA expression	TH	1	Regulate tumor progression	[178,184]
		PHOX2B	/	Be associated with familial NB	[180,185]
	Abnormal microRNA expression	MiR-124-3p	/	Inhibit NB invasion	[186,190]
cfDNA	Epigenetic alterations	RASSF1A methylation	1	Be associated with adverse outcomes	[121,197]

1 Biological changes observed in both tissue and liquid biopsies of NB.

2 Biological changes observed only in liquid biopsy in NB.

Combaret et al. report for the first time that MNA (MYCN amplification) status in NB and serum samples cfDNA is highly consistent across all disease stages [166]. In NB, Takahiro Gotoh et al. measured and analyzed the serum DNA of the MNA group and non-MNA group patients, which had been distinguished by Southern blotting, and found that the MYCN/NAGK ratio of the MNA group was significantly higher than that of the non-MNA group [167]. And they empirically identified MNA patients with an MYCN/NAGK ratio greater than 10. The results obtained by this method were in agreement with the Southern blot (100%). This suggests that measuring the MYCN/NAGK ratio of serum DNA can reliably detect MYCN amplification in NB. Several similar articles have reported the feasibility of using cfDNA to detect MYCN amplification [61,[168], [169], [170]]. A recent meta-analysis showed that MNA status in patients with advanced NB can be determined by MNA analysis of cfDNA [171]. MNA analysis of pooled cfDNA from eligible patients found an overall sensitivity of 0.908 (95% CI, 0.818-0.956) and specificity of 0.976 (0.940-0.991). A recent study demonstrated that improved detection techniques can accurately assess MYCN and ALK oncogene status in the cfDNA of small amounts of NB [172]. MYCN amplification is a common variant in NB, and recent experimental evidence suggests that it can contribute to enhanced neuroblastoma invasion by combining non-canonical enhancers and promoters [173].

These results demonstrate that genomic alterations in cfDNA and ctDNA are highly consistent with tissue DNA, and genomic alterations in cfDNA and ctDNA can reliably detect NB-specific biomarkers. And variants found only in ctDNA may represent tumor heterogeneity. This simultaneously demonstrates the feasibility and necessity of cfDNA and ctDNA to detect genomic alterations in NB. This means that in practical clinical applications, feasibility means that cfDNA and ctDNA are reliable adjuncts to detect genomic alterations. However, more studies are needed to demonstrate that variants found only in cfDNA and ctDNA are NB-specifically associated.

To date, few studies have detected genomic alterations in CTCs of NB. A recent study demonstrated that it is feasible to measure the DNA ploidy state of CTCs using flow cytometry [53]. Hyperdiploidy was detected using ISx at the first relapse in the NB case sample 33, whereas MYCN and ALK amplifications were later found on SNP arrays of cfDNA of BM (bone marrow) aspirates. These results demonstrate the reliability of CTC in detecting DNA ploidy. But this is far from enough to reveal the biological information about NB contained on CTC. Recently Masato Kojima et al. simulated the entry of CTCs into the blood and isolated them for single-cell sequencing [174]. They compared the sequencing depth and amplification efficiency of different sequencing methods, demonstrating the reliability of single-cell sequencing. Single-cell sequencing has been applied to other cancers [175], [176], [177], which is undoubtedly a great inspiration for revealing the genomic alterations contained in CTCs of NB.

3.2. Aberrant expression of the transcriptome

Detection of transcriptomic alterations in tumors can be performed in tumor tissue samples or tumor cell lines. The detection of transcriptome alterations in tumor cells in blood is rarely performed because of the influence of the transcriptome of other cells in the recipient fluid. Among cfRNAs, the abnormal expression of mRNA in NB has been a concern since the 1990s (Table 2) [178]. Limited by whether the mRNAs are derived from NB cells, studies have mainly focused on mRNAs specifically expressed in neurons, such as TH (tyrosine hydroxylase) [178], UCH-L1 (PGP9.5) [179], Doublecortin (DCX) and PHOX2B [143,179,180]. Y. Miyajima et al. pointed out that TH mRNA expression was detected in both NB tissue and blood, but not in normal control cells [178]. This indicates that the detection of TH mRNA in peripheral blood is highly feasible and specific. In the NB group of BM (bone marrow) or PB (peripheral blood), Janine et al. reliably demonstrated that the mRNA levels of PHOX2B, TH, DDC, CHRNA3, and GAP43 all showed high expression, while in the control group there was no or low expression [180]. Other studies have shown similar results [144]. In liquid biopsies of NB, only limited NB-specific mRNAs are currently targeted. In the future, with the maturity of single-cell RNA sequencing, it is hoped that a comprehensive mRNA expression that reflects tumor heterogeneity can be detected [174,[181], [182], [183]]. TH is an important substance that catalyzes the biosynthetic pathway of catecholamines involved in regulating the function of tumor progression (Table 2) [184]. PHOX2B mutations are frequently seen in familial NB cases and are also associated with the activation of the MAPK pathway (Table 2) [185,186].

In recent years, the role of microRNAs (miRNAs) responsible for the post-transcriptional regulation of NB has been of interest [187]. Matthew J Murray et al. were the first to reliably detect the overexpressed miR-124-3p, miR-218-5p, miR-9-3p, miR-1538, miR-490-5p in MNA serum of NB [141,188]. The expression of these miRNAs is elevated in MNA NBs compared to non-NB tumors and non-MNA NBs [141]. In another study on NB, miR-124-3p, miR-218-5p, and miR-490-5p were also expressed at high levels in serum [189]. MiR-124-3p has been shown to directly target and repress cytoskeletal genes associated with NB invasion (Table 2) [190]. A study found the overexpression of hsa-miR199a-3p in the plasma exosomes of NB patients [89]. These suggest that liquid biopsy can accurately and reliably measure the expression status of miRNAs in NB.

3.3. Epigenetic alterations

Epigenomics and epitranscriptomics have important biological roles [191,192], and abnormal changes in epigenetics are inseparable from tumors [193]. We separate a separate section to present epigenetic alterations found in liquid biopsies of NB. A Misawa et al. analyzed the tissue samples and blood samples of 68 neuroblastoma that compared the matching of 68 neuroblastoma [131]. They found that 94% of the tumor tissue sample discovered the methylation state of the RASSF1A gene, and 25%of serum samples observe the methylation of RASSF1A. These results indicate that the liquid biopsy can detect a part of the methylation state, but the sensitivity is not high. RASSF1A acts as a tumor suppressor gene, and its promoter hypermethylation silences its expression and promotes tumor progression [194]. Before this, Shigeki Yagyu et al. also proved the abnormal high methylation of Nb DCR2 promoters that can be identified by serum. A recent study suggests that high methylation RASSF1A can be used to mark NB ctDNA [195]. Liekel et al. reliably and sensitively identify high methylation RASSF1A in cfDNA of metastatic neuroblastoma [196]. RASSF1A methylation is associated with adverse outcomes in NB (Table 2) [197]. These results show that the abnormal methylation state of NB through liquid biopsy is feasible. As far as we know, no one currently evaluates the change of epitranscriptomics in the liquid biopsy of NB.

4. Clinical significance of liquid biopsy in NB

Above we described important biological alterations found in liquid biopsies of NB. The mechanism of these important biological changes in the occurrence and development of NB needs to be further explored. We focus on describing the clinical significance of these biological changes in NB.

4.1. Diagnosis

Studies are accumulating on the use of liquid biopsy in the diagnosis of NB (Fig. 3a, Table 3). Circulating tumor cells (CTCs) are often found in the blood when diagnosing patients with NB [54,72]. Detection of CTCs from NB can help to better non-invasively diagnose NB patients clinically. Copy number change and ALK mutation are important biomarkers in the diagnosis of NB [198,199]. We have described above that many studies have reliably detected copy number changes (MNA) and ALK mutations by liquid biopsy [60,61,156,[166], [167], [168], [169], [170], [171]]. Sho Kurihara et al. demonstrated that the amount of cfDNA was significantly correlated with the stage of NB [200]. We also described in the previous section the genetic heterogeneity of NB exhibited by liquid biopsies. For example, in plasma, blood, and tissue samples from some patients, common genomic changes (ALK mutation and MYCN amplification) were found only in plasma or blood samples [108,166]. Importantly, cfDNA epigenetics are associated with the early stages of tumors [201,202]. Circulating endothelial cells (CTECs) may be biomarkers for early diagnosis of early-stage NSCLC patients due to their association with tumor proliferation and migration [203]. These results have important significance for the noninvasive diagnosis of NB. Biopsy remains the gold standard in the diagnosis of NB. The use of liquid biopsy in the diagnosis of NB requires more preclinical and clinical studies.

Fig. 3.

Circulating biomarkers have important clinical implications in NB. (a) The number of cfDNA, copy number changes (MNA), ALK mutations, and epigenetics are important biomarkers for the diagnosis of primary NB. The appearance of CTCs correlates with tumor aggressiveness and is associated with both primary and recurrent NB diagnoses. MD is associated with NB recurrence. (b) The copy number changes (MNA), and RASSF1A methylation detected in cfDNA are important biomarkers for the prognosis of NB. The expression of cfDNA (CHGA, DCX, DDC, PHOX2B, and TH) was associated with NB prognosis. (c) Exosomal microRNAs (exo-miRNAs) can monitor treatment response, ctDNA levels have been shown to correlate with treatment response, and cfDNA can assess subclonal changes that emerge after treatment. (d) The expression of cfRNA can be detected for MRD monitoring, such as CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B, and TH these important biomarkers.

Table 3.

Application of liquid biopsy in NB. 

Application	Year	Method	The number of patients	Found	Citation
Diagnosis	2019	RT-qPCR	17	Increased expression of HSA-mir199a-3p in exosomes is associated with the severity of NB patients	[89]
	2016	OncoScan	70	The cfDNA may reflect genetic changes in more aggressive cell clones	[60]
	2017	WGS	37	The cfDNA analysis can easily detect copy number variations, such as MYCN and LIN28B amplification and ATRX deletions	[61]
	2015	dPCR	44	Preoperative abnormal mutations in cfDNA were detected, consistent with the original tumor test	[200]
	2002	qPCR	102	Circulating MYCN has been detected in patients with MNA NB	[166]
Assessment of prognosis, treatment	2000	Immunocytological quantitative analysis	466	Testing CTCs can identify patients with very high-risk diseases that are responsive to treatment	[72]
	2001	RT-PCR	49	Tyrosine hydroxylase mRNA level is associated with a poor prognosis of NB	[205]
	2005	RT-PCR	25	MYCN status and TH expression of circulating RNA remain important prognostic factors	[206]
	2017	qPCR	101	The mRNA levels of CHGA, DCX, DDC, PHOX2B, and TH were associated with progression-free survival (PFS)	[143]
	2011	qPCR, MLPA analysis	24	DNA fragments released by the tumor in the serum were examined for 11q loss	[117]
	2009	Methylation specific PCR	68	Serum RASSF1A methylation was statistically correlated with prognosis	[131]
	2008	qPCR	86	Methylated DCR2 was detected in serum DNA	[109]
	2013	qPCR	50	The copy number of MYCN was determined by the determination of circulating DNA	[211]
	2014	RT-qPCR	290	Higher mRNA levels in peripheral blood or bone marrow were associated with EFS and OS	[144]
Early diagnosis of relapse	1990	Immunocytological analysis	23	The presence of circulating neuroblasts was significantly correlated with tumor recurrence	[54]
	1992	RT-PCR	18	PGP 9.5 RT-PCR helps evaluate circulating tumor cells to assess early recurrence	[179]
	2011	RT-qPCR	102	MD in peripheral blood can predict disease progression and assess early relapse	[204]
Surveillance	2017	ddPCR	10	The cfDNA can reliably reflect MYCN and ALK status and improve monitoring of disease progression	[116]
	2019	RT-qPCR	52	Exosomal microRNAs can distinguish between adverse and beneficial reactions	[214]
	2018	WGS	10	Changes in ctDNA levels were associated with reversal of treatment	[107]
	2017	WES	19	The cfDNA analysis revealed small subclonal changes	[121]
Detection of minimal residual lesions	2009	RT-qPCR	56	The sensitivity of the five markers to different samples was different	[180]
	2013	RT-PCR	14	Eleven RT-QPCR markers could be used for more sensitive MRD monitoring in patients with NB	[222]
	2016	RT-qPCR	2	MRD monitoring in PB based on 11 RT-QPCR markers can detect the possibility of NB recurrence or regeneration	[219]
	2021	RT-qPCR	19	The correlation between MRD and tumor markers (VMA, HVA, NSE, and LDH) was limited	[224]

Relapse is associated with poor survival of NB, and evaluation of biomarkers associated with recurrence in NB may aid in the early diagnosis of relapse [54]. This may have important clinical significance for improving patient survival. Studies have demonstrated the potential of liquid biopsy in the early diagnosis of NB recurrence (Table 3). Moss et al. analyzed circulating tumor cells in the blood of patients with NB by hematological immunocytology and found that circulating tumor cells were associated with tumor relapse [54]. One experiment showed that PGP 9.5 RT-PCR could detect circulating NB cells with higher sensitivity than immunocytology [179]. The trial also found that one patient with circulating NB cell positive was at high risk for recurrence but had no evidence of other diseases [179]. A study evaluating peripheral blood microlesions (MD) in non-metastatic NB patients found that microlesions in stage 1 and 2 NB patients may not be associated with prognosis [204]. However, detection of MD in PB by QRT-PCR during diagnosis of stage 3 NB patients can predict disease progression and assess early recurrence. These results suggest that CTC and peripheral blood micropathology may be important markers for early detection and diagnosis of NB recurrence. However, more studies are needed to validate the use of liquid biopsy in the diagnosis of NB relapsed (Table 3).

4.2. Assessment of prognosis

Many studies have demonstrated that liquid biopsy can detect and identify adverse prognostic markers of NB [205,206] (Fig. 3b, Table 3). Detection of poor prognosis of NB is of great clinical value in risk stratification, treatment evaluation, and recurrence diagnosis [20]. One study examined tyrosine hydroxylase mRNA levels in children with NB by liquid biopsy and found that tyrosine hydroxylase mRNA levels were associated with a poor prognosis of NB [205]. This may be related to the spread of NB cells through the bloodstream. Andreu et al. examined the expression of tyrosine hydroxylase (TH) mRNA levels in peripheral blood of patients with NB [206]. And they found that TH expression in peripheral blood was associated with poor survival of poor prognosis. One experiment applied liquid biopsies in the blood of patients with NB to detect CHGA, DCX, DDC, PHOX2B, and TH mRNA levels and found that the mRNA levels of these genes were associated with progression-free survival (PFS) [143]. Copy number variation is an important prognostic factor for NB, such as MYCN amplification (MNA) and deletion of the long arm allele on chromosome 11 (11q deletion) [20,117]. The MYCN amplification status of circulating DNA in NB was assessed by real-time quantitative PCR analysis [167,168]. The sensitivity of the analysis is related to the tumor stage [168]. Liquid biopsies have been used to detect deletions of the long arm allele on chromosome 11 (11q deletions) in the circulating DNA of NB [117]. Copy number changes assessed by liquid biopsy tests can be applied with further prognostic assessment. The epigenetic inheritance of NB is related to the development of tumors [207], [208], [209]. One test assessed the methylation status of the RASSF1A gene in serum DNA from patients with NB by liquid biopsy. In this study, serum methylation of RASSF1A was statistically correlated with prognosis [131]. There have been tests using liquid biopsy to detect DCR2 methylation in serum DNA of NB [109]. The biopsy is expected to be a noninvasive method to predict the prognosis of NB. In a rare mechanistic study of NB by liquid biopsy, Jing Ma et al. found that abnormal upregulation of plasma exosomal hsa-miR199a-3p could promote the proliferation and migration of NB by regulating NEDD4 expression [89]. This suggests that exosome overexpression of hsa-miR199a-3p in liquid biopsy can be a biomarker for the treatment of NB.

Copy number change is an important biomarker for the risk stratification of NB [198]. For example, MYCN amplification is an important risk stratification indicator for NB [210]. And above we describe some studies that can reliably detect copy number changes by liquid biopsy. Masato et al. used quantitative real-time PCR (qPCR) to assess the copy number of MYCN in DNA from plasma of 50 NB patients and 10 healthy volunteers [211]. This experiment demonstrates that liquid biopsy can be used to detect MYCN amplification in NB. In addition, plasma MYCN copy number was associated with surgery and neoadjuvant chemotherapy. This suggests that liquid biopsy may be useful in evaluating the use of treatment in patients. A clinical trial used RT-QPCR to detect mRNA levels of PHOX2B, tyrosine hydroxylase (TH), and double cortisol (DCX) in peripheral blood and bone marrow biopsy fluid of children at high risk for NB [144]. The results show that high mRNA levels in peripheral blood or bone marrow at diagnosis were associated with poorer event-free survival (EFS) and overall survival (OS). Peripheral blood transfused high TH (log10TH>0.8) or high PHOX2B (log10PHOX2B>0.28) were identified as ultrahigh risk patients at diagnosis. After induction therapy, ultrahigh risk patients had worse event-free survival (EFS) and overall survival (OS) than other risk patients. The results suggest that assessing high levels of TH and PHOX2B mRNA in peripheral blood of patients with NB at diagnosis may help identify patients at high risk of having a very poor prognosis. In a clinical trial for the treatment of metastatic melanoma, patients with BRAF V600 mutations assessed by the same method were found to have higher tumor burden and lower PFS (progression-free survival) and OS (overall survival) [212]. Variations associated with poor prognosis found in other tumors by liquid biopsy provide an important clinical reference for the prognostic study of NB [213]. And liquid biopsy may provide a technical approach for developing new therapies for patients at high risk. Liquid biopsies will help assess patients at different risks for stratified therapy.

4.3. Surveillance of NB

Invasive biopsies of tumors are often not performed continuously, especially in children with NB. And tumors change from moment to moment. NB is highly heterogeneous. It is necessary to monitor NB dynamically. Current imaging methods are used to evaluate and monitor high-risk NB [214]. Increasing use of liquid biopsies to detect molecular disease signatures associated with NB surveillance (Fig. 3c, Tables 2 and 3). A study using xenotransplantation trials and retrospective trials showed that liquid biopsies can accurately assess MYCN and ALK copy number status of primary NB by droplet digital PCR (ddPCR) protocol [116]. This provides a basis for the application of liquid biopsy in the monitoring of disease progression. Martina et al. found that liquid biopsy could distinguish between adverse and favorable responders by detecting the characteristics of exosome microRNAs (exo-miRNAs) in NB blood [214]. In recent years, circulating tumor DNA (ctDNA) levels have been found to be correlated with treatment response [215,216]. Kelly et al. used ultra-low passage whole genome sequencing to detect and evaluate ctDNA in the blood of patients with NB [107]. Ultra-low passage whole genome sequencing is a new liquid biopsy analysis method developed by them. They found that changes in the levels of ctDNA collected at different time points were associated with treatment reversals. A clinical study (NCT02856893) determined response to treatment with osimertinib versus gefitinib in non-small cell lung cancer (NSCLC) progression by plasma ctDNA detection of EGFR T790M, but results were not available [58]. These studies indicate the important value and possibility of liquid biopsy for the detection of neuroblasts. During the development of NB, there may be new mutations that lead to therapeutic resistant cloning, which we call clonal evolution. A study comparing cfDNA assessments in NB blood at two different time points found small subclones change [121]. These results suggest that liquid biopsies can aid in the detection of resistant mutated clones.

4.4. Detection of minimal residual lesions

The detection of minimal residual disease (MRD) in tumors by liquid biopsy has demonstrated its clinical significance in many tumors [217]. The presence of residual malignant cells in normal cells after intensive treatment of high-risk NB is called minimal residual disease (MRD) [218]. Activation of minimal residual disease is closely related to the prognosis of NB [219]. More than 50% of high-risk NB patients will experience recurrence or tumor growth as a result [219]. In the current study, RT-qPCR is often used to evaluate minimal residual disease of NB. PHOX2B is a common RT-qPCR marker for MRD detection (Fig. 3d) [220]. PHOX2B was shown to be more specific and sensitive than TH and GD2 synthases in the detection of MRD in patients with NB [221]. A study of markers for the detection of minimal residual lesions in patients with NB has demonstrated the potential of combining PHOX2B, MRDTH, DDC, CHRNA3, and GAP43 markers to detect samples with different sensitivities [180]. One study showed that 11 RT-qPCR markers (CHRNA3, CRMP1, DBH, DCX, DDC, GABRB3, GAP43, ISL1, KIF1A, PHOX2B, and TH) can be used to achieve more sensitive MRD monitoring in patients with NB [222]. RT-qPCR based on these markers has been reported to monitor the dynamics of MRD in patients with NB to assess treatment response and disease status [223]. In recent years, the clinical significance of MRD monitoring based on liquid biopsy is being investigated. One study showed that MRD monitoring based on the above 11 RT-qPCR markers detected the possibility of NB recurrence or regrowth [219]. A recent trial examined the association of MRD with tumor markers (VMA, HVA, NSE, and LDH) [224]. The results showed limited correlation. These results suggest that the detection, monitoring, and evaluation of minimal residual disease (MRD) based on liquid biopsy has important potential clinical value in NB (Table 3). More studies are needed for clinical evaluation.

5. Prospects and challenges

Studies on the application of liquid biopsy in the diagnosis, treatment stratification, prognosis assessment, and disease surveillance of NB are reviewed. Research on the utility of liquid biopsy in the diagnosis of NB is increasing (Fig. 3). Liquid biopsy may play an important role in the early screening of NB. Cancer screening is essential, although many people believe that only finding cancer markers is beneficial. Early screening for cancer requires simplicity, low cost, low impact on the human body, high specificity, and sensitivity. Moreover, liquid biopsy detection is simple, has high accuracy, and has no effect on the human body. More studies are needed to confirm its feasibility. Liquid biopsy has unique advantages in NB surveillance. Liquid biopsy has unique advantages in NB surveillance. The Liquid biopsy allows noninvasive continuous testing of NB to monitor treatment response. Especially in children at high risk of NB, other continuous invasive or ionizing radiation detection methods are difficult to implement clinically. The rapidity of liquid biopsies may not be as good as imaging techniques, but liquid biopsy is relatively safe. Liquid biopsy monitoring of children with NB can observe the changes of biomarkers after treatment to reflect the treatment effect and adjust the treatment strategy. The monitoring of liquid biopsy can dynamically observe the disease status and adjust the treatment strategy if the disease burden increases. In recent years, there have been many studies on liquid biopsy in detecting minimal residual disease of NB. In recent years, there have been many studies on liquid biopsy in detecting minimal residual disease of NB. The clinical significance of liquid biopsy in detecting minimal residual disease remains to be further clarified. The study of minimal residual disease detected by liquid biopsy and other adverse biomarkers is also an important direction. At the same time, liquid biopsy is an important method to evaluate prognosis. More novel prognostic biomarkers will be evaluated by liquid biopsies in the future (Fig. 4).

Fig. 4.

Timeline of studies related to liquid biopsy of NB. More and more studies have been done on the application of liquid biopsy in NB. Here are a few important studies from 1990 to 2021. In recent years, the liquid biopsy of NB is mainly used to analyze circulating nucleic acid (circulating DNA and circulating RNA), and the study of minimal residual disease is a hot topic of current research.

The International NB Risk Group (INRG) working group has recommended that PB samples be taken from patients with NB at diagnosis, before and after myeloablative treatment, and at the end of treatment. And PB will be analyzed by standard methods to facilitate the evaluation of the clinical significance of MD and MRD [225]. This indicates that the liquid biopsy regimen has been applied to the clinical practice of NB. However, the clinical application of liquid biopsy for NB requires more prospective trials. But that requires universal support. This is still a long way off, but research is accumulating. More clinical trials will be conducted in the future to advance the clinical application of NB. The current requirements for liquid biopsy technology, equipment, and facilities are also important issues. Whether analytical techniques can be applied universally remains a question. Many laboratories and medical facilities have limited facilities and facilities for liquid biopsy analysis, which can affect the quality and results of the overall test. These are also the questions that should be considered now.

Immunotherapies for various cancers are currently developing rapidly, including immune checkpoint inhibitors [67]. However, resistance to immunotherapy can also occur [226]. inhibitors (ICIs) show no efficacy in epidermal growth factor receptor (EGFR) and HER2-altered cancers. At this time, the detection of biomarkers in the immunotherapy stage is required to improve the treatment plan. The only current first-line immunotherapy that requires diagnostic testing of biomarkers is the assessment by tissue biopsy [67]. Compared with tissue biopsy, liquid biopsy has a greater advantage in immunotherapy. Liquid biopsy can be better done with more limited invasiveness because of the ease of obtaining valid samples for biomarker assessment during treatment. Recent studies have found that mice with melanoma xenografts can release PD-L1-bearing exosomes into the bloodstream [227]. This increase in exosomal PD-L1 has been shown to be associated with poorer prognosis in melanoma patients [227]. In conclusion, liquid biopsy shows great potential for clinical application in immunotherapy, which requires further development of detection technology and research of liquid biopsy.

Liquid biopsy has become an emerging adjuvant method of attention compared to tissue biopsy because of some of its advantages. First, one of the great advantages of liquid biopsy is that it can theoretically reflect tumor heterogeneity. Compared with tissue biopsy, liquid biopsy can better reflect the biological characteristics of different tumor cells. In principle, tissue biopsies detect only local tumor cells, ignoring the high heterogeneity of NBs. In addition, the entry of substances such as NB cells into the blood may also reflect their adverse disease progression. This is undoubtedly an important tool in the study of metastatic and high-risk NB. Second, the non-invasive means of liquid biopsy provides an adjunct to patients. Especially in pediatric cancer treatment evaluation, invasive tissue biopsy limits our evaluation during treatment, while non-invasive liquid biopsy can provide an adjunct to better clinical treatment evaluation. Also, current analyses of liquid biopsies are not limited to assessing circulating tumor cells but include DNA, RNA, and proteins. This has greatly facilitated the development of analytical techniques based on genomics, transcriptomics, and epigenetics. Currently, there are few studies on circulating tumor proteins. It is worth mentioning that some biological characteristics of drug-resistant and persistent subclones can be detected in the detection and monitoring of NB by liquid biopsy. This is undoubtedly an important adjunct to the personalized treatment of NB.

Compared with tissue biopsy and medical imaging, above we described the huge advantages of liquid biopsy in the diagnosis, monitoring, etc. of early primary and recurrent tumors. However, liquid biopsies still have significant limitations. First of all, ctDNA is currently identified by the biomarkers related to NB that have been discovered, which is not without loopholes in theory. It is difficult to ensure that DNA derived from other somatic cells is free of relevant mutations. This also limits the diagnostic utility of ctDNA. Second, one liquid biopsy is always limited. It has been suggested to combine different assays to better diagnose and manage cancer patients, which is undoubtedly the best option for cancer patients [228]. For example, tumor-specific methylation tends to be present in early-stage tumors, but not as much in early-stage ctDNA [229,230]. Therefore, some studies have proposed that the combination of ctDNA and methylation detection can improve the reliability of diagnosis. Finally, liquid biopsy is currently not a substitute for tissue biopsy. Although liquid biopsies have shown great promise in tumor monitoring and treatment evaluation, liquid biopsies have a long way to go in the clinical setting of NB. Overall, the clinical application of liquid biopsy into NB and even other cancers is promising.

The continuous development of liquid biopsy technology will promote the application of liquid biopsy in NB. More research is needed to gain general support. On this basis, continuous preclinical and clinical experiments are needed to promote the clinical application of liquid biopsy in NB and even other tumors. Clinical application of liquid biopsy in NB is imminent.

Author contributions

Zhenjian Zhuo, Lei Lin, and Lei Miao collected the related literature Lei Lin, Lei Miao, and Meng Li wrote the manuscript. Zhenjian Zhuo and Jing He participated in the design of the review and revised the manuscript. All authors have read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no conflicts of interest in this work.

Biographies

Zhenjian Zhuo received his Ph.D. degree from the Chinese University of Hong Kong. His postdoctoral training was done at Guangzhou Women and Children's Medical Center. Now he works as an associate researcher at Peking University Shenzhen Graduate School. His current research interests focus on cancer genetics, cancer epidemiology, and pediatric cancers. He is the associate editor for Cancer Medicine, Journal of Oncology, and Heliyon.

Jing He is a principal investigator of Guangzhou Women and Children's Medical Center. He received his Ph.D. degree from Fudan University. His research interests include molecular epidemiology on cancers especially on pediatric cancer, such as neuroblastoma, Wilms tumor and hepatoblastoma. He has published more than 200 scientific research articles, with an H index of more than 34.