Patients
A total of 116 high risk NB patients were recruited in the Hematology Oncology Center, Beijing Children’s Hospital from February 1, 2015 to December 31, 2017. Informed consent was obtained from parents or guardians of each patient according to the Declaration of Helsinki. High risk neuroblastoma was classified as age older than 18 months, and stage IV by International Neuroblastoma Staging System (INSS); or any age, and stage II, III or IV with MYCN gene amplification. All patients had received multiple-disciplinary treatment, then went into maintenance treatment after evaluation. All patients were monitored and evaluated during maintenance treatment. The whole follow-up ended at September 30, 2018. This research and the BCH-NB-2007-HR protocol were approved by the Beijing Children’s Hospital Institutional Ethics Committee (No. 2016-65). The BCH-NB-2007-HR protocol was based on the Hong Kong Pediatric Hematology and Oncology Study Group (HKPHOSG) [21] and a German report.[22]
Diagnostics and Evaluation
For initially diagnosed patients, microscopic examinations of bone marrow aspirates and biopsies were performed to identify the presence of neuroblastoma cells. Genetic abnormalities, i.e., amplification of the MYCN gene, deletion of the short arm of chromosome 1 (1p36) and the long arm of chromosome 11 (11q23), were detected by Fluorescence in situ hybridization. Serum tumor markers such as lactate dehydrogenase (LDH), neuron specific enolase (NSE), and cfDNA were quantified.
The evaluation of therapeutic response was performed, when multiple-disciplinary treatment finished, by serum tumor markers quantification, minimal residual disease (MRD) detection including 131I- metaiodobenzylguanidine (131I- MIBG), ultrasound and computed tomography (CT). According to the standards of Response Evaluation Criteria in Solid Tumors (RECIST), therapeutic response was classified into four degrees, complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD). Patients of CR, PR and SD could step into maintenance treatment.
Evaluation during maintenance treatment included serum tumor markers detection, microscopic examinations of bone marrow and imaging examination every three months, and 131I- MIBG every six months.
Treatment
According to the BCH-NB-2007-HR protocol, initially diagnosed high risk NB patients received multiple-disciplinary treatment including induction chemotherapy, surgery, consolidation therapy, and radiotherapy. Some patients received autologous stem cell transplantation. Regular regimens included chemotherapy with high dose cyclophosphamide, adriamycin and vincristine (CAV), chemotherapy with high dose cisplatinum and VP16 (CVP), surgery after 4-5 cycles of chemotherapy, and then harvest peripheral blood stem cell for possible autologous hematopoietic stem-cell rescue.
Maintenance treatment was with 13-cis-retinoic acid 160 mg/m2/day, 14 days on alternative with 14 days off for 9-12 months; and 13-cis-retinoic acid stopped.
Sample collecting
Blood samples were collected to quantify cell-free DNA (cfDNA) at time points of the beginning of maintenance treatment, every three months afterwards, and diagnosis of recurrence. Venous blood samples were collected in EDTA-coated tubes and plasma was separated by centrifugation at 1600g for 10 minutes. Supernatant was transferred into a new tube and centrifuged at 16000g for 10 minutes. Purified plasma was carefully removed from the tube and stored at -80°C for DNA extraction.
Plasma cfDNA detection
Before quantification, DNA was extracted from 200 μL plasma and eluted by 300 μL elution buffer by QIAmp DNA Blood Mini Kits (Qiagen, Valencia, CA, USA). cfDNA level detection was detailed in our previous paper.[23] Briefly, after preparation of extracted DNA, quantitative polymerase chain reaction (qPCR) was performed on LightCycler LC 480 PCR (Roche Molecular Systems, Inc, Pleasanton, CA, USA) to amplify the apoptotic and nonapoptotic LINE-1 79bp DNA fragments by specific primers. A reference standard curve was established by serially diluting standard solution of human genomic DNA (Thermo Fisher Scientific, Waltham, Ma, USA). The qPCR reaction mixture contained 2 μL of eluted DNA, 1 μL (final concentration 0.2 μm) of each forward and reverse primer of LINE-1 79bp, 5 μL of UltraSYBR Mixture (Cwbiotech, Beijing, China), and 1 μL of double-distilled water. Cycling conditions were 1 minute at 95°C and 35 cycles of 95°C for 8 seconds and 60°C for 15 seconds. The qPCR reaction was performed in triplicate and the mean of the triplicates were used for further analysis. Each plate contained as a plasma DNA sample, a water template (negative control) and 7 serially diluted standard DNA solutions. The cfDNA level of each sample was detected based on standard curve by using the 2-△△Ct method.
Statistics analysis
Statistical analysis was performed in R statistical environment (version 3.4.0), including Mann-Whitney U test, Chi-Square test, and ROC (receiver operating characteristic, Bioconductor ROC package). A p-value lower than 0.05 was considered statistically significant.