To evaluate plasma cell‐free DNA (cfDNA) as a promising biomarker for neuroblastoma (NB) tumor burden. Seventy‐nine eligible patients with newly diagnosed NB were recruited from Beijing Children's Hospital between April 2016 and April 2017. Additionally, from September 2011 to June 2017, 79 patients with stable NB were evaluated with a median follow‐up time of 21 months. Approximately 2 mL of peripheral blood was drawn upon enrollment, and plasma cfDNA levels were measured via quantitative polymerase chain reaction (qPCR). Total cfDNA analysis was performed using the long interspersed nuclear element 1 (LINE‐1) 79 bp fragment, and DNA integrity was calculated by the ratio of the LINE‐1 300 bp fragment to the LINE‐1 79 bp fragment. A total of 79 NB patients with a median age of 36 months comprised the group of newly diagnosed NB patients. The main primary tumor site was the retroperitoneal and adrenal region (81%). Three or more metastatic sites were found in 17.7% of patients. Stable NB patients older than 18 months comprised 98.7% of the stable NB patients. Neuron‐specific enolase (NSE), lactate dehydrogenase (LDH), and cfDNA levels were dramatically increased in the newly diagnosed NB patients and significantly different from those in the stable NB patients. Moreover, the concentration of cfDNA was much higher in patients with larger tumors. By analyzing the area under the receiver operator characteristic (ROC) curve (AUC), the areas of total cfDNA, NSE, and LDH levels were 0.953, 0.929, and 0.906, respectively. The sensitivity and specificity data clarified that the level of circulating cfDNA in plasma can be considered as a reliable biomarker for describing tumor load in NB. The plasma cfDNA concentration was as good as the levels of LDH and NSE to discriminate the tumor burden in children with NB.

MYCN gene amplification is related to risk stratification. Therefore it is important to identify accurately the level of the MYCN gene as early as possible in neuroblastoma (NB); however, for patients with bone marrow (BM) metastasis who need chemotherapy before surgery, timely detection of the MYCN gene is not possible due to the unavailability of primary tumors. MYCN gene status was evaluated in 81 BM metastases of NB by interphase fluorescence in situ hybridization (FISH) analysis of BM cells. The clinicobiological characteristics and prognostic impact of MYCN amplification in NB metastatic to BM were analyzed. MYCN amplification was found in 16% of patients with metastases, and the results were consistent with the primary tumors detected by pathological tissue FISH. MYCN amplification was associated with age, lactate dehydrogenase (LDH) levels and prognosis (P = 0.038, P < 0.001, P = 0.026). Clinical outcome was poorer in patients with MYCN amplification than in those without amplification (3-year EFS 28.8 ± 13.1 vs. 69.7 ± 5.7%, P = 0.005; 3-year OS 41.5 ± 14.7 vs. 76.7 ± 5.5%, P = 0.005). MYCN amplification predicts a poor outcome in NB metastatic to BM, and interphase FISH of bone marrow cells provides a timely direct and valid method to evaluate the MYCN gene status.

Importance: Neuroblastoma is the most common extracranial malignant solid tumor in children. Multidisciplinary care is critical to improving the survival of pediatric patients with neuroblastoma.Objective: To systematically summarize the clinical characteristics of children with neuroblastoma and evaluate their prognosis with multidisciplinary care provided in a single center.Methods: This retrospective study analyzed the clinical data of 1041 patients with neuroblastoma who were diagnosed, treated, and followed‐up in the Hematology‐Oncology Center of Beijing Children’s Hospital from 2007 to 2019.Results: The median age at diagnosis was 34 months; 80.8% of the patients were younger than 5 years of age. Notably, 243 patients (23.3%) were classified as low‐risk, 249 patients (23.9%) were classified as intermediate‐risk, and 549 (52.7%) were classified as high‐risk. Furthermore, 956 patients underwent surgical resections; 986 (94.7%) patients received chemotherapy; and 176 patients with high‐risk neuroblastoma received hematopoietic stem cell transplantation. The 5‐year event‐free survival (EFS) rate was 91.3% and 5‐year overall survival (OS) rate was 97.5% in low‐risk group; in the intermediate‐risk group, these rates were 85.1% and 96.7%, respectively, while they were 37.7% and 48.9% in the high‐risk group (P < 0.001 for both). The 5‐year EFS and OS rates were significantly higher in patients diagnosed between 2015 and 2019 than in patients diagnosed between 2007 and 2014 (P < 0.001). In total, 278 patients (26.7%) exhibited tumor relapse or progression; the median interval until relapse or progression was 14 months. Of the 233 patients who died, 83% died of relapse or progression of neuroblastoma and 4.3% died of therapy‐related complications.Interpretation: The 5‐year OS rate was low in high‐risk patients, compared with low‐and intermediate‐risk patients. Multidisciplinary care is critical for improvement of survival in pediatric patients with neuroblastoma. Additional treatment strategies should be sought to improve the prognosis of patients with high‐risk neuroblastoma.

We ought to explore the acquired somatic alterations, shedding light on genetic basis of somatic alterations in NB patients with chemotherapy. Marrow blood samples from NB patients were collected before treatment, after the 2nd and 4th chemotherapy for baseline research and continuous monitoring by whole exome sequencing. Plasma cell free DNA (cfDNA) was prepared for baseline research. Finger nail cells were extracted as self control. The clinical data was analyzed. From December 2014 to February 2016, 27 cases of children with stage IV NB were diagnosed. The follow up time ranged from 5 to 25 months, with a median follow up time of 17 months, 20 patients were stable, one patient died of pulmonary embolism during surgery, six patients died of disease progression. Marrow blood whole exome sequencing demonstrated that several novel somatic mutations were identified in all three trios comply or against the trendy of tumor size variation. Of note, six recurrent mutations in bromodomain PHD finger transcription factor (BPTF) were identified in nine NB patients under the continuous monitoring. The mutation rates variation was positively correlated to tumor size (CC = 0.428, P = 0.021), and patients with BPTF mutation may have a worse prognosis compared with wild type. Meanwhile, CGREF1, CUX2, GP1BA, SLC45A1 and TRA2A were mutated with the trendy oppose as therapeutic effects. The baseline research in three NB patients demonstrated that mutation rate of BPTF, TMCO3, GPRIN2 and C20orf96 in plasma cfDNA were in positive correlation with bone marrow genomic DNA (P = 0.001). Our study showed that BPTF along with other mutations may function as a biomarker for evaluating to effects of chemotherapy to this refractory tumor, and patients with BPTF mutation might have a worse prognosis.

Neuroblastoma (NB) is the most common extracranial solid tumor and the treatment efficacy of high-risk NB is unsatisfactory. γδT-cell-based adoptive cell transfer is a promising approach for high-risk NB treatment. Our previous study has revealed that γδT cells in NB patients exhibit a poor proliferation activity and a decreased anti-tumor capacity in vitro. In the present study, we found that IL-15 could effectively enhance the proliferation of NB γδT cells, to a level that remains lower than healthy controls though. In addition, IL-15-fostered NB γδT cells robustly boosted cell survival against apoptosis induced by cytokines depletion. Our data revealed that Mcl-1 was a key anti-apoptotic protein in IL-15-fostered γδT cells during cytokine withdrawal and its expression was regulated via the activation of STAT5 and ERK. In addition, IL-2 and IL-15-fostered γδT cells harbored higher levels of tumoricidal capacity which is also beneficial for γδ T-cell based immune therapy in NB. Understanding the survival control of γδT cells in a sub-optimal cytokine supportive microenvironment will expedite the clinical application of γδT cells for immunotherapy.

Background: Neuroblastoma is the most common extracranial solid tumor of childhood. The high rate of recurrence is associated with a low survival rate for patients with high-risk neuroblastoma. There is thus an urgent need to identify effective predictive biomarkers of disease recurrence. Methods: A total of 116 patients with high-risk neuroblastoma were recruited at Beijing Children’s Hospital between February 2015 and December 2017. All patients received multidisciplinary treatment, were evaluated for the therapeutic response, and then initiated on maintenance treatment. Blood samples were collected at the beginning of maintenance treatment, every 3 months thereafter, and at the time of disease recurrence. Plasma levels of cell-free DNA (cfDNA) were quantified by qPCR. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the ability of plasma cfDNA concentration to predict recurrence. Results: Of the 116 patients, 36 (31.0%) developed recurrence during maintenance treatment. The median time to recurrence was 19.00, 9.00, and 8.00 months for patients who had achieved complete response (n = 6), partial response (n = 25), and stable disease (n = 5), respectively, after multidisciplinary treatment. The median plasma cfDNA concentration at the time of recurrence was significantly higher than the concentration in recurrence-free patients throughout maintenance treatment (29.34 ng/mL vs 10.32 ng/mL). Patients recorded a plasma cfDNA level ≥ 29 ng/mL an average of 0.55 months before diagnosis of disease recurrence. ROC analysis of the power of plasma cfDNA to distinguish between patients with or without recurrence yielded an area under the curve of 0.825, with optimal sensitivity and specificity of 80.6 and 71.3%, respectively, at a cfDNA level of 12.93 ng/mL. Conclusions: High plasma cfDNA concentration is a potential molecular marker to signal disease recurrence in patients with high-risk neuroblastoma.