Plasma circulating cell‐free MYCN gene: A noninvasive and prominent recurrence monitoring indicator of neuroblastoma

Abstract The postoperative recurrence of neuroblastoma (NB) patients is an essential reason for the high mortality of NB due to the lack of early, non‐invasive, and dynamic strategies for monitoring NB recurrence. Therefore, whether the plasma circulating cell‐free MYCN gene as an indicator for monitoring of NB recurrence was systematically evaluated. The MYCN copy number and NAGK (reference gene) copy number (M/N) ratio in plasma and corresponding tumor tissues of NB patients was detected using an economical, sensitive, and specific single‐tube dual RT‐PCR approach developed in this study. The plasma M/N ratio of the MYCN gene amplification (MNA) group (N = 25, median M/N ratio = 4.90) was significantly higher than that of the non‐MNA group (N = 71, median M/N ratio = 1.22), p < .001. The M/N ratio in NB plasma (N = 60) was positively correlated with the M/N ratio in NB tumor tissue (N = 60), with a correlation coefficient of 0.9496. In particular, the results of dynamic monitoring of postoperative plasma M/N ratio of NB patients showed that an abnormal increase in M/N ratio could be detected 1–2 months before recurrence in NB patients. In summary, the single‐tube double RT‐PCR approach can be used to quantitatively detect MYCN copy number. The copy number of MYCN in the tissue and plasma of NB patients is consistent with each other. More importantly, the circulating cell‐free MYCN gene of NB patients can be used as a monitoring indicator for early, non‐invasive, and dynamic monitoring of NB recurrence.

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 highrisk 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][7][8][9][10][11][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 is a lack of clinical methods for the early, noninvasive 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][14][15] The overexpression of MYCN inducing transcriptional activation of MYCN, increased MYCN protein stability due to dysregulated MYCN phosphorylation, and reduced proteasome degradation to MYCN gene amplification is closely related to the progression of NB. [16][17][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][21][22][23][24][25][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 in non-MNA NB. [27][28][29][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 plasma MYCN 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 MYCN copy 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 ratio, a highly sensitive and specific single-tube multiplex RT-PCR approach was developed using a MYCN molecular 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 MYCN copy 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.

| Reagents, materials and instruments
All DNA used in this study was purchased from Sangon Biotech (Shanghai, China) and all sequences are listed in Table S1. Diethyl pyrocarbonate (DEPC) treated water, and dNTP were purchased

| Sample preparation
Tumor specimens were surgically removed and immediately stored in liquid nitrogen. To avoid contamination of plasma DNA by white blood cell (WBC) DNA, whole blood of patients with NB was centrifuged at 3000 r/min for 10 min within 2 h, plasma was separated and stored at À80 C until DNA extraction.

| Cell culture and DNA Isolation
SK-N-BE2, SK-N-AS, SH-SY5Y and HUVEC cell lines were cultured in DMEM supplemented with 10% FBS, penicillin (100 μg/ml) and streptomycin (100 μg/ml) in 5% CO 2 at 37 C. Total extraction kit DP304 purchased from Tiangen Biotech (Beijing China) was used to extract the total DNA in cell lines NB tissue samples and plasma specimens.

| Single tube duplex RT-PCR
The single tube duplex RT-PCR was conducted by the following   x + 36.583 (y and x are the C T value and NAGK copy number, respectively) and the correlation coefficient is R 2 = 0.9970. Figure 1F shows the real-time fluorescence curve of different NAGK copy numbers where the C T value increases with a decrease in NAGK copy number.
On the logarithmic scale, C T values and NAGK copy numbers yielded a good linear relationship on seven orders of magnitude from 10 to 10 7 copies. The correlation equation is y = À3.473 lg x + 36.56 (y and x are the C T value and NAGK copy number, respectively) and the correlation coefficient is R 2 = 0.9967. Therefore, the developed singletube duplex RT-PCR can quantitatively detect MYCN and NAGK in a single tube with high sensitivity. SH-SY5Y and HUVEC is less than 2 ( Figure 2A). Furthermore, the amplification products of MYCN and NAGK in NB tissues were sequenced based on the single-tube double RT-PCR method and the sequencing results were more than 99% similar to the target sequences ( Figure 2B,C). Therefore, the single-tube double RT-PCR method established in this study has high specificity in real sample analysis. In addition, the reproducibility of the developed single-tube duplex RT-PCR recipe was investigated by five consecutive assays where the RSDs for MYCN and NAGK were determined to be 1.3% and 0.7%, respectively ( Figure S3). The results show that the developed single-tube duplex RT-PCR recipe has acceptable reproducibility.

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In summary, the developed approach has high specificity in real sample analysis and acceptable reproducibility.

| The M/N ratio in NB tumor tissue and plasma
The total DNA was extracted from 96 cases of NB plasma samples and 60 of cases NB clinical samples of NB tumor tissues, and the MYCN copy number in plasma and tissues. The OD 260 /OD 280 of total DNA extracted from plasma and tissue specimens were detected with a range from 1.80 to 1.90, which indicated that the extracted DNA was of good quality (Table S2) There were no statistically significant differences between the MNA group and the non-MNA group in terms of gender, age, and stage of diagnosis (Table 1) Experimental results showed that the correlation coefficient between plasma M/N ratio and tumor tissue M/N ratio was 0.9496 ( Figure 3C).
Therefore, the M/N ratios in NB plasma and tumor tissues are consistent with each other.

| Dynamic detection of M/N ratio in NB plasma
The

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The research proposal was approved by the ethics review committee of Children's Hospital Affiliated to Zhengzhou University.