Cancer involves the uncontrolled division of cells with immortal proliferative capacity. The tumor cells acquire genomic instability and an increasing number of mutations during each division, which allows them to evade cell cycle control, escape immune destruction, and form a suitable “niche” microenvironment with increased vasculature. Finally, the acquisition of invasive and metastatic properties leads to cancer becoming a systemic disease with serious clinical consequences. In addition to advances in early detection, surgical resection, chemotherapy and/or radiotherapy strategies, more recent developments involve targeted therapies based on comprehensive analysis of gene mutations [1].

The incidence of neuroblastoma (NB) is 11–13 cases per million children aged 14 years. In children aged 12 months, the incidence of NB is 65 cases per million according to SEER databases. Survival from NB is much better in younger children [2], and this cancer type is very rare in adulthood.

NB is a neural embryonal cancer of early childhood that arises from sympathetic ganglion cells derived from neural crest cells. These cells migrate from the neural tube during early embryogenesis [3, 4, 5, 6]. In children, NB originates from the adrenal medulla or from cells of the sympathetic nervous system on both sides of the spine (paravertebral ganglia). Palpable swelling in the abdomen, significant bruising around the eyes, and various other clinical signs and symptoms can be observed depending on the origin of NB. When NB develops on both sides of the spine, it can cross the vertebrae and apply pressure to the spinal cord, leading to conditions such as loss of strength or complete paralysis in the legs, as well as urinary or fecal incontinence. NB shows considerable heterogeneity in clinical, pathologic and molecular properties. To assess its tumor burden, biology and prognosis, NB is categorized into very low, low, intermediate, high and very high risk categories. Patient age, tumor stage (L1, L2, M, and MS), surgical risk factors, imaging results, grade of tumor differentiation, and biomarkers (N-Myc amplification, 17q gain, 1p deletion, 11q deletion, DNA ploidy) are used in risk assessment [7, 8]. The International Neuroblastoma Risk Group (INRG) staging system was first proposed by Cohn and Pearson and updated by the Children’s Oncology Group (COG) [9, 10, 11, 12].

Many genetic and epigenetic events occur during neural crest maturation and migration from the neural tube to primordial neuroectoderm, and then to mature ganglia or adrenal medulla. These include genetic alterations to ZIC1, PAX3, TPAP2a, Notch, PRDM1A, SOXE family genes, ZEB2, BMP, Wnt, FGF, FOXD3, C-Myc, and N-Myc. Gain of 17q, loss of 1p, and loss of 11q are the most common genetic changes, while N-Myc amplification (20–25% frequency) is a biomarker for advanced disease. Functional molecular oncology approaches have recently been used in an attempt to link clinicopathologic properties with specific molecular changes [13, 14, 15, 16].

The various therapies for NB include surgical removal of the tumor mass, chemotherapy, radiotherapy, immunotherapy, and autologous stem cell transplantation. Despite some success in managing this disease, approximately half of NB cases are high-risk and the 5-year survival rate is 40%. Multi-agent chemotherapy has been used in high-risk cases [17, 18]. Chemotherapy combinations are also used for relapsed cases, some of which become refractory to treatment and hence the patient becomes prone to disseminated metastatic disease. In such cases, immunotherapy and or targeted therapies may improve patient survival.

The number of targeted drugs approved by the FDA is quite limited, especially for NB and pediatric tumors. Treatments targeted to 43 gene changes were approved in 28 different tumor types, most of which are adult cancers. Current targeted therapy trials for high-risk NB include the Anaplastic Lymphoma Kinase (ALK) inhibitors Crizotinib and Lorlatinib. The R1275Q mutation in ALK is sensitive to first generation ALK inhibitors, whereas patients with F1174 mutations are resistant. However, patients with F1174 mutations are responsive to second generation ALK inhibitors [19, 20]. The aurora kinase inhibitor Alisertib (MLN8237) is also currently being tested for NB in combination with chemotherapy. This drug indirectly disrupts N-Myc to induce cell cycle arrest, apoptosis, and autophagy. Other agents being tested for NB include the Raf inhibitor Sorlafenib, PI3K inhibitors that block the RAS pathway, and the anti-VEGF bevacizumab [13]. Immunotherapy with the monoclonal anti-disialoganglioside GD-2 has also been shown to improve the survival of high-risk NB patients [10]. The most difficult scenario encountered during the treatment of cancer patients is the lack of response to any treatment. In such cases, the detection of a targetable mutation may provide the final treatment opportunity. Health insurance companies do not usually cover this treatment, although permission to use off-label drugs can be requested.

Most targeted therapy agents require molecular assessment to provide evidence for indication. The detection of somatic variants in tumor tissue by high throughput sequencing has been used in clinical practice to identify predictive markers, as well as for diagnostic or prognostic purposes. The investigation of some molecular targets is case-specific, for example the assessment of mutation status for a specific targeted therapy according to cancer and organ subtype. Some targeted therapy inhibitors are agnostic, such as the neurotrophic tyrosine kinase (NTRK) inhibitors. Hence, the detection in any tumor of NTRK fusion leading to increased tyrosine kinase activity provides indication for the use of NTRK inhibitors.

Next-generation sequencing (NGS) allows the detection of rare individual gene mutations [21]. Although somatic mutations are infrequent in NB, they have been observed in a few genes such as ALK [22, 23]. NB is generally referred to as a “cold” tumor type because it has a very low mutation burden. The NGS method is now used routinely for molecular evaluation of solid tumors in adults. It is also increasingly being used for pediatric cancers [24, 25]. cDNA is obtained from RNA by reverse transcriptase and then sequenced by NGS. This method is becoming widely used, especially for the evaluation of translocations. Although still expensive, RNA analysis for translocations is especially important for diagnosis and for the selection of targeted therapy in soft tissue, central nervous system and salivary gland tumors [26].

Each year about 3000 cases of childhood cancer are diagnosed in the Turkish Republic, of which around 300 are NB. Half of these NB cases are metastatic at diagnosis. Increased knowledge of NB at the molecular level is needed to better understand its pathogenesis and to develop novel therapeutics that could improve outcomes in high-risk, relapsed, or refractory cases. The aim of the present study was therefore to present targeted NGS data for somatic mutations in a Turkish cohort of relapsed and refractory NB cases.

Targeted, capture-based NGS was performed on 60 selected cancer-related genes. This was designed to explore targeted therapy options for relapsed and refractory NB patients. DNA was extracted from tumor tissues that were freshly frozen and stored at –80 °C, or from formalin-fixed and paraffin-embedded (FFPE) tumor tissue from diagnostic archival tissue. RNA was also extracted and converted to complementary DNA for the detection of 87 fusions in 18 genes.

In the present study we report NGS mutation and fusion data for relapsed and refractory NB patients from our institute. We found several potential treatment targets, notably ALK mutations, fusions in NTRK, ROS1 and RET, BRAF mutations other than V600E, the common ERBB2 I655V mutation, as well as other rare mutations for which the clinical relevance in NB has yet to be established.

The ERBB2 I655V missense mutation was common in this NB cohort. ERBB2 is located at 17q12 and is a well-known receptor tyrosine kinase involved in growth factor signaling. Clinical trials targeting ERBB2 are ongoing with agents such as fam-trastuzumab deruxtecan in non-small cell lung cancer and breast cancer. The only pathogenic mutation found in ERBB2 in our NB cohort was I655V. ERBB2 is altered in 5.44% of malignant solid tumors, including adenocarcinomas of the gastroesophageal junction (19.62%), urothelial carcinomas (14.87%), breast carcinomas (13.78%), gastric adenocarcinoma (10.08%), cervical carcinoma (9.09%), malignant uterine neoplasm (8.03%), colorectal carcinoma (4.69%), non-squamous non-small cell lung carcinoma (4.28%), non-small cell lung carcinoma (3.97%), biliary tract carcinoma (5.41%), glioblastoma (1.84%), WHO grade III glioma (1.55%), and some diffuse midline glioma, H3 K27M-mutant patients. ERBB2 I655V mutation is an inclusion criterion in open clinical trials (mainly phase 2) for malignant solid tumors [37].

EGFR signaling is important for cell growth and proliferation in high-risk NB, and copy number gains that span ERBB2 are common. High levels of ERBB2 expression or amplification are used routinely to select HER2-positive breast cancer patients for targeted therapy with trastuzumab. ERBB2 is reported to be altered in 2.4% of NB patients and to date there has been only one clinical trial. ERBB2 I654 and I655V mutations are genetic risk factors for cancer [37].

Most NB cases with ALK mutations have N-Myc amplification [38]. Half the cases with ALK mutations in the present series also had N-Myc amplification. One of the observations in the present study that should be confirmed in a larger series is that familial cases with germline R1275Q mutations did not show N-Myc amplification.

Mutations in the FGFR pathway are observed in 7% of all cancers. Small molecule drugs are available that target FGFR, including pemigatinib, infigratinib, erdafitinib, derazantinib and futibatinib. Ongoing clinical trials in cholangiocarcinoma and metastatic urothelial carcinoma have shown encouraging results for FGFR inhibitors, either alone or in combination with immunotherapy. FGFR2 fusions or rearrangements, or loss of function mutations in the FGFR2 gene (FGFR2 D530N and FGFR2 A648T) could therefore be used for targeted therapy. In the present NB cohort we found FGFR3 (e17):TACC3 (e11) fusion and one case with FGFR1 D133 duplication. Disruption of FGFR activity could also change the NB microenvironment in terms of tumor-associated fibroblasts or macrophages, as well as immune cells such as T lymphocytes [39, 40].

In addition to guiding targeted therapy, NGS results can be used for diagnosis and prognosis in various cancer types. The sequencing panel used in the present study does not provide much information for diagnosis, but the prognostic value of detected mutations should be explored in larger patient series. Anti-FGFR targeted therapies for NB should also be explored with regard to possible side-effects and tumor resistance.

The BRAF proto-oncogene is involved in the mitogen-activated protein kinase (MAPK) signaling pathway. The BRAF V600E mutation is considered to be a class I mutation type and is commonly found in melanoma, colorectal cancer and multiple myeloma, but is very rare in NB. New types of mutations (class II and class III) have now been defined in cancer, including aberrant transcriptional gene regulation. Class II mutations include non-V600E mutations in the RAS binding domain, RAS wild type, and RAS independent kinase activated cases. Class III mutations include the kinase domain, RAS-dependent, and RAS mutant cases [41]. We speculate the rare BRAF mutations observed in NB tissue with one or more RAS mutations could be classified as Class III mutations. These mutations are located near the P-loop (G466). The prognostic and therapeutic role of BRAF mutations that coexist with KRAS mutations in NB require further investigation, together with the importance of the BRAF L588P mutation in the kinase domain.

Despite the small number of cases in our patient cohort, we found similar mutations to those reported in other recent studies of relapsing, therapy-resistant NB. A limitation of the present work is that the 60-gene panel does not include some genes related to NB, including N-Myc. NGS cannot replace Fluorescence In Situ Hybridization (FISH) or PCR for the detection of Segmental Chromosomal Aberrations (SCA). For example, Kim et al. [42] recently compared NGS and FISH for SCA of 1p deletion, 11q deletion and 17q gain in 35 NB samples. They concluded that NGS was a sensitive complementary and alternative method to conventional FISH for detecting SCAs in NB [42]. However, the NGS method is not yet accepted as a gold standard for the detection of SCA in NB. We are currently comparing the FISH, real-time PCR, digital PCR, and NGS methods in our laboratory for routine testing of N-Myc amplification and 11q deletion. The cost of NGS increases when SCA is added to the panel. It is preferable to use 8 DNA control samples to compare losses or gains of sequenced genes.

In general, routine NGS analysis is critical for the clinical management of patients with solid tumors. The use of NGS for pediatric cancers is also gaining widespread use. An experienced team that includes pathologists familiar with molecular techniques, molecular biologists/oncologists, bioinformaticians, and clinical oncologists is necessary to obtain and correctly interpret NGS data. Appropriate preanalytical procedures including good tumor tissue selection and storage are also very important. One of the challenges is how to report a rare mutation detected by the bioinformatic analysis that is not linked to a clinically proven indication for targeted therapy. The multidisciplinary team should be aware of the function of the relevant gene and whether targeted drugs exist for its protein.

We believe the importance of this research is that genes related to targeted therapy, even more than the known and approved molecular changes, may offer new opportunities for treatment in relapsed/ refractory NB patients. For successful molecular evaluation it is important to use methods that work well with low quality and/or low quantity tumor tissues, including bone marrow tru-cut biopsies that have undergone decalcification. Whole exome sequencing generally requires larger amounts of sample DNA. Targeted somatic exome sequencing using CLIA-certified NGS platforms such as MSK-IMPACT or Foundation One NGS panels may provide other choices that avoid whole genome sequencing [43]. The whole exome comprises 1.5% of the entire genome, including the coding regions [44].

Samoyedny et al. [45] reported the adequacy of NGS was 88% in a study of 95 cases of NB-positive biopsies. Their series included image-guided biopsies for relapsed NB. NGS adequacy was also reported to be 82% for bone tumor biopsies and 96% for soft tissue tumor biopsies [45]. In the present NB series, an NGS adequacy of 100% was achieved through a step-by-step evaluation of the preanalytical quality and quantity of sample. We prefer primary biopsy or surgical tissue material obtained prior to the use of neoadjuvant therapy. Starting with fresh frozen tissue or FFPE, we select blastic tumor areas without necrosis, hemorrhage, or normal tissue. If this is not available, we use lymph node, soft tissue, and bone marrow biopsies that contain metastatic tissue, and macrodissect the tumor blastic tissue with high purity into a microtube. If the quantity is low following DNA and RNA extraction, the extraction procedure is repeated from the beginning.

From the perspective of bench to bedside, the rare mutations detected in this study may lead to targeted therapy trials in relapsed and/or refractory NB patients. These mutations may offer clues for the development of new targeted drugs, or for improving existing targeted drugs so they can act on the known target as well as the rare loci. In future, we will continue NGS molecular evaluation of NB cases by improving the panel, in parallel with the International NB study group reports. We also plan to use NGS for all cases at diagnosis and to routinely test for N-Myc amplification, 11q24 deletion and possibly DNA ploidy, together with other panels related to targeted therapy. The future NGS plans for our Molecular Oncology Laboratory will consider the latest reports from the College of American Pathologists, the Association of Molecular Pathology, and the Catalog of Somatic Mutations of Cancer database [46, 47].

For cases where tumor tissue is not available, we have also started an NGS liquid biopsy panel. We will examine liquid biopsy in a prospective study by detecting DNA in plasma for the follow-up of minimal residual disease in NB. Successful NGS using liquid biopsy has been reported for many cancer types including NB [48], although fewer selected genes are studied with this sample type. For example, a panel of 8 oncogenes (EGFR, KRAS, BRAF, HER2, MET, ALK, RET, and ROS1) was used to study non-small cell lung cancer [49]. Multicancer liquid biopsy panels are also sold commercially, but none are yet available for NB or pediatric cancers. Customized panels could be designed, or a commercial panel suitable for pediatric cancers could be used, such as the multi-cancer cfDNA panels for sequencing more than 30 genes. Ideally, a customized panel for cfDNA and designed for NB would include the mutations found in the present study and in the literature.

Pugh et al. [50] used whole exome sequencing to search for mutations in paired samples of NB tissue and peripheral blood. They found a low somatic mutation burden that included mutations in ALK (9.2%), PTPN11 (2.9%), ATRX (2.5), MYCN (1.7%), and NRAS (0.83%). Their NB series was significantly enriched for pathogenic germline variants in ALK, CHEK2, PINK1, and BARD1 [50]. In the present series we only searched for germline mutations when familial NB was suspected clinically. This was a limitation of our study, but the main aim was to investigate mutations related to targeted therapy. Another limitation of this study was that important NB-associated mutations such as ATRX and MYCN may have been missed due to the targeted NGS of selected genes from a cancer panel. However, this ready-to-use panel results in far fewer NGS failures. The panel also includes genes for which targeted therapy drugs are already available.

In conclusion, the NB cases in this study showed mainly ALK F1174L (sensitive to alectinib), and R1275Q (sensitive to crizotinib) mutations. Rare mutations were also found in the tyrosine kinase domain. EGFR, ERBB2, BRAF, ABL1, MET and PIK3CA mutations were also found in NB, but their clinical importance remains to be determined. This study supports the detection by NGS of SNV mutations as a useful tool to identify predictive, diagnostic and prognostic markers in NB. The use of methods and validated kits that are suitable for individual molecular oncology laboratories is necessary. Appropriate panel selection is also important. An important challenge is to obtain good results with low quality and low-input tissues. Although infrequent, fusions should also be routinely investigated in NB patients to identify additional opportunities for targeted therapy.

The data might be shared upon request anonymously after ethics approval.

TA, SA, NO designed the research study. DK, DI, EC, NO, EO collected data and patients. TA, SA, ES, AE, OG, SMO, ZA performed the research and laboratory work. TA, SA analysed the data. TA, SA, OG, NO wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.

Ethical Approval was taken from Dokuz Eylul University Non-invasive Research Ethics Committee (14.12.2022, No: 2022/40-03). Informed Consent was signed to parents of the patients.

We thank to Dokuz Eylul University Rectorate for the support of the study. The data might be shared upon request anonymously after ethics approval.

This study was supported by Dokuz Eylul University Scientific Research Councill (Project no: TSA-2023-3007).

The authors declare no conflict of interest.