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.
A dual isothermal amplification assay with dual fluorescence signal detection strategy, named dual isothermal amplification all-in-one approach, was developed for rapid, one-step, highly sensitive quantification of plasma circulating MYCN copy number of neuroblastoma (NB). The developed strategy consisted of rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP) on a real-time PCR system using highly specific probe, molecular beacon (MB), as detection probe. The developed strategy possessing a broad linear dynamic range of 10 aM to 1 pM for both target gene ( MYCN ) and reference gene ( NAGK ) . The ratio of the MYCN copy number to NAGK copy number (M/N ratio) was detected by the developed approach in cell lines, NB tumor tissues, hepatoblastoma tumor tissues and Wilms’ tumor tissues, to which the M/N ratios were consistent with previous reports. In particular, the M/N ratio in NB clinical tissue specimens and NB plasma specimens detected with the developed approach were in keeping with the standard RT-PCR approach. More importantly, the M/N ratio in NB tissue samples and corresponding plasma samples of NB patients were consistent with each other with a correlation coefficient of 0.9690, indicating that plasma circulating MYCN is a promising indicator for the risk classification of NB.
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