Can Qi Zi w. Zhao Lin Chen Le Wang Yun Zhou Guochen Duan ^*

• Hebei Provincial Children's Hospital, Shijiazhuang, Hebei Province, China

Background: Cancer cells induce neutrophil extracellular traps (NETs) to promote tumor progression and metastasis. However, only a few studies have focused on the role of NETs in Neuroblastoma (NB).Methods: First, based on the expression of NET-related genes, consensus clustering analysis was conducted to cluster NB samples into different subtypes. Differential analysis was performed to identify DEGs between subtypes. Functional items and related pathways of DEGs were identified using enrichment analysis. Univariate Cox analysis and the LASSO algorithm were used to identify biomarkers for prognosis. Furthermore, independent prognostic analysis was performed. Immune infiltration analysis was performed to identify differential immune cells. Finally, the verification of prognostic model genes were taken by the immunohistochemical staining and quantitative real-time PCR.Results: Consensus clustering analysis demonstrated that NB samples were clustered into two subtypes. There were 125 DEGs between the two subtypes of NB. Moreover, the enrichment analysis results showed that the DEGs were mainly associated with 'external side of plasma membrane,' 'immune receptor activity' 'regulation of leukocyte migration' GO items. KEGG pathways revealed that the DEGs were correlated with in immunity-related activities, including 'Complement and coagulation cascades,' 'Neutrophil extracellular trap formation, 'T cell receptor signaling pathway,' 'PD-L1 expression and PD-1 checkpoint pathway in cancer' and so on. A total of five biomarkers,[ Selenoprotein P1 (SEPP1), Fibrinogen-like protein 2 (FGL2), NK cell lectin-like receptor K1 (KLRK1), ATP-binding cassette transporters 6(ABCA6) and Galectins(GAL)], were screened, and a risk model based on the biomarkers was created. Furthermore, a nomogram for forecasting the survival rates of patients with NB was established based on the risk score, age at diagnosis, and MYCN status. Eight differential immune cells were acquired between the two risk subgroups. The expression levels of five prognostic model genes at the protein and mRNA were verified and all results were consistent with the results of our bioinformatics analysis.Conclusion:We initially found that five NET-related genes were significantly differentially expressed in NETs-associated molecular isoforms and two Netrg molecular isoforms were found to be associated with poorer prognosis. This stratification might provide insight into the prediction of prognosis and ideal immunotherapy strategies for patients with NB.