The aim of this study was to investigate the prognostic role of MYCN RNA expression by quantitative RNA in situ hybridization and its association with MYCN amplification in neuroblastoma. MYCN RNA expression in 69 neuroblastoma tumors was evaluated by an ultrasensitive quantitative RNA in situ hybridization technique, RNAscope. The correlations between MYCN RNA expression, MYCN amplification, and other clinicopathologic variables of neuroblastoma were analyzed. High expression levels of MYCN RNA were detected 30 of 69 (43%) of neuroblastomas, mainly in those with undifferentiated or poorly differentiated histology. High expression of MYCN RNA was significantly associated with MYCN amplification (P < 0.001) and other adversely prognostic factors, including older age at diagnosis (>18 months, P = 0.017), advanced clinical stage (International Neuroblastoma Staging System stage 3, 4, P = 0.002), unfavorable International Neuroblastoma Pathology Classification tumor histology (P < 0.001), and high-risk Children’s Oncology Group risk group (P = 0.001). In Kaplan–Meier analysis, MYCN RNA levels determined by quantitative in situ hybridization were better than MYCN gene dosages determined by chromogenic in situ hybridization in discriminating good and poor prognostic groups of neuroblastoma patients. In multivariate analysis, we further confirmed that high expression of MYCN RNA was an independent adverse prognostic factor for event-free and overall survival. Furthermore, high expression of MYCN RNA predicted unfavorable survival outcomes for neuroblastoma patients with MYCN non-amplification or high-risk Children’s Oncology Group risk group. In conclusion, our study is the first report to show the application of MYCN RNA in situ hybridization in neuroblastoma and established that high expression of MYCN RNA could be a better biomarker than MYCN amplification for predicting poor prognosis of neuroblastoma patients.

本研究的目的是通过定量 RNA 原位杂交研究MYCN RNA 表达的预后作用及其与神经母细胞瘤中MYCN扩增的关联。通过超灵敏定量 RNA 原位杂交技术 RNAscope 评估了 69 例神经母细胞瘤中的MYCN RNA 表达。分析了MYCN RNA 表达、MYCN扩增和神经母细胞瘤的其他临床病理变量之间的相关性。69 个神经母细胞瘤中有 30 个（43%）检测到MYCN RNA的高表达水平，主要是在那些组织学未分化或分化差的神经母细胞瘤中。MYCN高表达RNA 与MYCN扩增 ( P  < 0.001) 和其他不良预后因素显着相关，包括诊断时年龄较大（>18 个月， P  = 0.017）、临床分期较晚（国际神经母细胞瘤分期系统第 3、4 期，P  = 0.002） ，不利国际神经母细胞瘤病理学分类肿瘤组织学（P  <0.001），和高危儿童肿瘤组危险组（P  =0.001）。在 Kaplan-Meier 分析中，通过定量原位杂交确定的MYCN RNA 水平优于MYCN通过显色原位杂交确定的基因剂量在区分神经母细胞瘤患者的良好和不良预后组中。在多变量分析中，我们进一步证实MYCN RNA 的高表达是无事件生存和总生存的独立不良预后因素。此外， MYCN RNA的高表达预示着MYCN非扩增或高风险儿童肿瘤组风险组的神经母细胞瘤患者的不利生存结果。总之，我们的研究是第一份显示MYCN RNA 原位杂交在神经母细胞瘤中应用的报告，并确定MYCN RNA 的高表达可能是比MYCN更好的生物标志物用于预测神经母细胞瘤患者预后不良的扩增。