Identification of New Gene Spliceosomes in Neuroblastoma

Neuroblastoma is an early childhood embryonic malignancy that originates from neural crest cells. Neuroblastoma shows high heterogeneity in biological, morphological, genetic, and clinical features. At present, the main treatment methods for neuroblastoma are surgical treatment combined with chemotherapy after the operation and immunological therapy. However， clinical studies have found that 40%～50% of patients don't have good outcomes after postoperative chemotherapy.

The clinical trial study aims to screen the genotype of children with neuroblastoma and conduct an in-depth analysis of sequencing data and tumor-specific transcripts by using technologies--Deep Sequencing Technology and Third Generation Sequencing Technology.

The main questions this study aims to answer are： \[Question 1\]This study will use Third Generation Sequencing technology to find specific transcript variants associated with cancer differentiation; \[Question 2\]This study will identify possible tissue differential expression by using the Deep Sequencing Technology.

Participants will undergo surgery, during which doctors will remove tumor tissue and adjacent normal tissue. In this experiment, the tumor tissue of children with neuroblastoma will be used as the experimental group, and the adjacent normal tissue will be used as the control group. In this study， the different genotypes of children with neuroblastoma are screened by Deep Sequencing Technology and Third Generation Sequencing Technology. And according to the difference in genotypes， doctors will treat children with neuroblastoma personally. This study hopes to find new single nucleotide polymorphism and therapeutic targets.

The clinical trial study aims to screen the genotype of children with neuroblastoma and conduct an in-depth analysis of sequencing data and tumor-specific transcripts using technologies--Deep Sequencing Technology and Third Generation Sequencing Technology. This study hopes to find new single nucleotide polymorphism and therapeutic targets.

Firstly, researchers collect tumor tissues and adjacent normal tissues of 20 children with neuroblastoma meeting the inclusion criteria and collect their clinical data. In this experiment, the tumor tissue of children with neuroblastoma will be used as the experimental group, and the adjacent normal tissue will be used as the control group.

Secondly, researchers will find differentially expressed genes using Deep Sequencing technology and Third Generation Sequencing Technology to sequence 7 neuroblastoma cell lines. The expression of the transcription isoforms was divided into one group of N-myc gene amplification and another group of N-myc gene non-amplification by using Third Generation Sequencing Technology. After extracting the total RNA from tissues, researchers check the RNA's purity, concentration, and integrity. If the RNA is tested up to standard, researchers will build a gene library. After the construction of the gene library is completed, researchers will use the machine to sequence tissues.

Finally, survival analysis and repeated measurements will be used to analyze the data of the experimental group and control group in this experiment. T-test will be used for measurement data. χ2 test will be used for counting data. Kaplan-Meier method and Cox regression analysis will be used for survival analysis. All statistical analyses will be performed using Statistical Analysis Software (Stata 26.0). P \< 0.05 is considered significant.

Eligibility Criteria:
Inclusion Criteria:

* Children with neuroblastoma range in age from 0 to 14, regardless of gender;
* Preoperative imaging examinations indicate patient with neuroblastoma;
* Before postoperative chemotherapy, the patient's physical strength is good. At the same time, the results of white blood cells, neutrophils, hemoglobin, platelets, and other test indicators are all within normal range, which is in line with the treatment conditions of the relevant chemotherapy and immunotherapy;
* Preoperative pathological examination of the patient reveals neuroblastoma;
* Patients have no other history of malignant tumors;
* Patients voluntarily participate and sign informed consent and can comply with the study visit plan and other protocol requirements.

Exclusion Criteria:

* Patients who are found to have distant metastasis or ascites on preoperative examination are excluded;
* Patients with severe liver and kidney dysfunction, as well as those with autoimmune diseases are excluded;
* Patients with severe cardiovascular disease who can not tolerate general anesthesia are excluded;
* Patients who have other malignancies or blood disorders are excluded.