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.