Patients
A total of 116 patients with high-risk NB were recruited at the Hematology Oncology Center, Beijing Children’s Hospital between February 1 2015 and December 31 2017. High-risk NB was classified as (i) age older than 18 months and stage IV disease according to the International Neuroblastoma Staging System (INSS); or (ii) any age and stage II–IV disease with N-Myc (MYCN) gene amplification. All patients had received multidisciplinary treatment, had been evaluated, and were then started on maintenance treatment. The patients were monitored and evaluated throughout maintenance treatment, with follow-up ending on September 30, 2018. This study and the BCH-NB-2007-HR protocol were approved by the Beijing Children’s Hospital Institutional Ethics Committee (No. 2016-65). Informed consent was obtained from the patients’ parents or guardians. The BCH-NB-2007-HR protocol is based on the Hong Kong Pediatric Hematology and Oncology Study Group guidelines [21] and the results of a study in Germany [22].
Diagnostic tests and Evaluation
Upon initial diagnosis, bone marrow biopsies and/or aspirates were obtained for microscopic examination and identification of NB cells. Genetic abnormalities (amplification of the MYCN gene, deletion of the short arm of chromosome 1 [1p36], and/or deletion of the long arm of chromosome 11 [11q23], were detected by fluorescence in situ hybridization. Serum levels of tumor markers, including lactate dehydrogenase (LDH) and neuron-specific enolase (NSE), were quantified.
After multidisciplinary treatment, the therapeutic response was determined by quantification of serum tumor markers, microscopic examination of bone marrow samples, 131I- metaiodobenzylguanidine (131I-MIBG) scanning, ultrasound, and computed tomography. According to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria, the response was classified as complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD). Patients with CR, PR, or SD entered maintenance treatment.
Quantification of serum tumor markers, microscopic examination of bone marrow, and imaging tests were performed every 3 months, and 131I-MIBG scanning was performed every 6 months.
Treatment
According to the BCH-NB-2007-HR protocol, patients initially diagnosed with high-risk NB received multidisciplinary treatment including induction chemotherapy, surgery, consolidation therapy, and radiotherapy. Some patients received autologous stem-cell transplantation. Common regimens included chemotherapy with high dose cyclophosphamide, adriamycin, and vincristine, chemotherapy with high dose cisplatinum and VP16, surgery after 4–5 cycles of chemotherapy, and harvesting of peripheral blood stem cells for possible autologous hematopoietic stem-cell rescue. The maintenance treatment regimen was 13-cis-retinoic acid 160 mg/m2/day on alternate days for 14 days followed by 14 days off treatment for 6–9 months.
Sample collecting
Blood samples were collected to quantify cfDNA at the beginning of maintenance treatment, every 3 months thereafter, and at the diagnosis of recurrence. Venous blood samples were collected into ethylenediaminetetraacetic acid-coated tubes and centrifuged at 1600 ×g for 10 min. Supernatants were transferred to fresh tubes and centrifuged at 16,000 ×g for 10 min. Plasma was removed and stored at −80°C until DNA extraction.
Plasma cfDNA detection
DNA was extracted from 200 µL plasma and eluted in 300 µL elution buffer using QIAmp DNA Blood Mini Kits (Qiagen, Valencia, CA, USA). cfDNA was quantified as previously described [23]. Briefly, DNA was subjected to quantitative polymerase chain reaction (qPCR) using a LightCycler LC 480 PCR (Roche Molecular Systems, Pleasanton, CA, USA). Primers were designed to amplify 79-bp fragments of long interspersed nuclear element 1 (LINE-1) DNA, which is derived from apoptotic and non-apoptotic cells. A reference standard curve was established with serial dilutions a standard solution of human genomic DNA (Thermo Fisher Scientific, Waltham, MA, USA). The qPCR reaction mixture contained 2 µL of eluted DNA, 1 µL each of forward and reverse LINE-1 79 bp primers (final concentration 0.2 µm), 5 µL of UltraSYBR Mixture (ConWin Biotech, Beijing, China), and 1 µL of double-distilled water. Cycling conditions were 1 min at 95°C and 35 cycles of 95°C for 8 s and 60°C for 15s. qPCR reactions were performed in triplicate and the mean value was used in calculations. Negative and positive controls (water as template and standard DNA dilutions) were included on each plate. cfDNA concentration was calculated from the standard curve using the 2-△△Ct method.
Statistics analysis
Data are presented as the median or mean and standard deviation and were analyzed using the Mann–Whitney U test or Chi-square test in R statistical environment (version 3.4.0). Receiver operating characteristic (ROC) curves were constructed and analyzed using the Bioconductor ROC package. A p value of <0.05 was considered significant.