1. Introduction
Neuroblastoma (NB), originating from the postganglionic sympathetic nervous system, is the most common extracranial malignant tumor in children. NB accounts for 8-10% of pediatric cancer cases and 15% of pediatric tumor deaths [1-3]. NB can be stratified into low-, intermediate-, and high-risk groups based on the demarcation of age at diagnosis, the International Neuroblastoma Staging System (INSS) stage, the tumor tissue MYCN status, the International Neuroblastoma Pathology Committee (INPC) classification, and ploidy [4]. Clinically, most NB patients, especially high-risk NB, are only diagnosed at an advanced stage due to the poor verbal ability of young NB patients, the hidden location of the tumor and ambiguous early symptoms of NB[5]. The 5-year overall survival rate of low-risk NB and intermediate-risk NB ranges from 85% to 90%. However, despite intensive multimode therapy used to treat high-risk NB over the past 30 years, more than 50% of high-risk NB patients still relapse, resulting in a 5 year survival rate of less than 10% with a long-term survival rate of only 2% [6-12]. Hence, early diagnosis for the recurrence of high-risk NB patients is one of the more effective ways to reduce the mortality of high-risk NB patients. However, there are a lack of clinical methods for the early, non-invasive and dynamic monitoring of recurrence in NB patients. Therefore, it is urgent to establish a non-invasive and dynamic detection strategy to monitor the recurrence of NB.
The NB tumor tissue MYCN gene (MYCN ) is a widely used clinical biomarker in NB risk grading. MYCN gene amplification (MNA) exists in 20-30% of NB patients, and the overall survival rate in these patients remains below 50% [13-15]. The overexpression ofMYCN inducing transcriptional activation of MYCN , increased MYCN protein stability due to dysregulated MYCNphosphorylation, and reduced proteasome degradation to MYCN gene amplification is closely related to the progression of NB [16-18]. In high-risk NB patients, MYCN amplification, if it occurs, is always present at diagnosis. NB patients with low-risk disease who lack MNA do not develop high-risk disease and do not acquire additional copies of MYCN gene [19]. This suggests that MNA is an early and possibly initiating event that drives the progression of high-risk NB. At present, the status of MNA is determined using either southern blots or fluorescence in situ hybridization (FISH) based on invasive tumor tissue samples. However, these methods are invasive, time-consuming and expensive and also require a relatively large amount of tumor tissue. In particular, NB is highly heterogeneous, which may result in deviation of test results and is not representative of the overall tumor phenotype [20-26]. Therefore, it is necessary to establish a non-invasive, rapid, sensitive and specific diagnostic method of MYCN status for the early diagnosis and recurrence detection of NB. Iehara, Ma, Combaret and Gotoh, have shown that plasma circulating cell-free MYCN in MNA NB patients is higher than non-MNA NB [27-30]. However, these studies did not conduct the following experiments: (1) the MYCN copy number in NB tumor tissue was not detected quantitatively; (2) Whether the plasmaMYCN copy number can dynamically monitor the NB recurrence was not systematically studied. Therefore, the MYCN copy number in NB plasma and tumor tissue was systematically examined in this study.
In order to accurately quantify the MYCN copy number in plasma and tumor tissue of NB patients, the N-acetylglucosamine kinase gene (NAGK was selected as the internal reference gene. NAGK is located on the same chromosome as MYCN but is sufficiently distanced from the MYCN amplicon region. The ratio of MYCNcopy number to NAGK copy number (MYCN / NAGK , M/N) was used to assess the amplification of MYCN copy number. To further accurately quantify M/N value, a highly sensitive and specific single-tube multiplex RT-PCR approach was developed using a MYCNmolecular beacon (MB) and NAGK MB to detect MYCN PCR products and NAGK PCR amplification products, respectively. Subsequently, the plasma and tumor tissue of NB was systematically studied using the developed single-tube multiplex RT-PCR approach. In particular, we dynamically monitored postoperative plasma MYCNcopy number in NB patients to further evaluate the feasibility of plasma circulating cell-free MYCN as a noninvasive indicator of NB recurrence using the developed single-tube multiplex RT-PCR approach. The innovations of this study are summarized as follows. (1) A highly sensitive and specific single-tube multiplex RT-PCR approach was developed to detect the plasma and tumor tissue M/N ratio. (2) The consistency of M/N ratio in plasma and tissue of NB patients was systematically evaluated. (3) The feasibility of using M/N ratio in plasma of MNA NB patients for non-invasive and dynamic monitoring of recurrence in NB patients was studied. This study is expected to provide theoretical support for early and non-invasive recurrence monitoring of MNA NB.