石蜡包埋组织中端粒酶表达的快速检测：原位杂交与定量 PCR 的联合应用。
Rapid detection of telomerase expression of neuroblastoma in paraffin-embedded tissue: combination of in situ hybridisation and quantitative PCR.
机构信息
Department of Pathology, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China.
Department of Surgical Oncology, Children's Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China.
出版信息
Pathology. 2023 Dec;55(7):958-965. doi: 10.1016/j.pathol.2023.07.005. Epub 2023 Sep 6.
Neuroblastoma is a heterogeneous paediatric malignant tumour. Telomere maintenance mechanism (TMM) by telomerase activation or alternative lengthening of telomeres (ALT) is a hallmark of high-risk neuroblastoma. However, the prior assays for telomerase, such as TERT expression by RNA sequencing or microarrays, may not be easy to perform in many histopathology laboratories in hospitals. The aims of this study are to assess the utility of ultrasensitive single-cell RNA in situ hybridisation (RNAscope), immunohistochemistry, and RT-qPCR on formalin-fixed, paraffin-embedded tumour samples as diagnostic tools for detecting TERT expression in neuroblastoma. In this study, we detected MYCN amplification in 22 of 222 cases (10%), TERT rearrangements in 18 of 220 cases (8%), and ALT activation in 39 of 222 cases (18%) using fluorescence in situ hybridisation (FISH). By RNA in situ hybridisation, 36 of 210 (17%) pretreatment neuroblastomas were found to have TERT overexpression, which was significantly associated with the high-risk group (33/78, 42%), TERT rearrangements (16/18, 89%), and MYCN amplification (13/22, 59%). None of the tumours with ALT showed TERT staining. In our study, 19 of the 55 MYCN non-amplified high-risk neuroblastomas displayed TERT mRNA expression, including 13 of the 14 TERT rearrangements, none of the 30 ALT-positive cases, and a significant proportion (6/11, 55%) that did not have the aforementioned genomic anomalies. RT-qPCR results correlated well with RNAscope levels (Spearman's rho=0.621, p<0.001, n=94). In conclusion, TERT RNA in situ hybridisation and RT-qPCR are suitable methods to evaluate TERT expression in neuroblastoma. The combination of detection of the genomic alterations and TERT mRNA expression is a powerful strategy for TMM activation detection, which can categorise neuroblastomas into multiple clinical subgroups for risk stratification in routine histopathology practice.
神经母细胞瘤是一种异质性小儿恶性肿瘤。端粒维持机制（TMM）通过端粒酶激活或端粒的替代性延长（ALT）是高危神经母细胞瘤的标志。然而，先前的端粒酶检测方法，如通过 RNA 测序或微阵列检测 TERT 表达，在许多医院的组织病理学实验室中可能不容易进行。本研究旨在评估超敏单细胞 RNA 原位杂交（RNAscope）、免疫组织化学和 RT-qPCR 在福尔马林固定、石蜡包埋肿瘤样本中的应用，作为检测神经母细胞瘤中 TERT 表达的诊断工具。在这项研究中，我们使用荧光原位杂交（FISH）检测到 222 例病例中的 22 例（10%）存在 MYCN 扩增，220 例病例中的 18 例（8%）存在 TERT 重排，222 例病例中的 39 例（18%）存在 ALT 激活。通过 RNA 原位杂交，发现 210 例（17%）预处理神经母细胞瘤中有 36 例存在 TERT 过表达，这与高危组（33/78，42%）、TERT 重排（16/18，89%）和 MYCN 扩增（13/22，59%）显著相关。没有一个 ALT 阳性的肿瘤显示 TERT 染色。在我们的研究中，55 例非 MYCN 扩增的高危神经母细胞瘤中有 19 例显示 TERT mRNA 表达，包括 13 例 TERT 重排、30 例 ALT 阳性病例中无一例、以及相当大比例（6/11，55%）没有上述基因组异常。RT-qPCR 结果与 RNAscope 水平高度相关（Spearman's rho=0.621，p<0.001，n=94）。总之，TERT RNA 原位杂交和 RT-qPCR 是评估神经母细胞瘤中 TERT 表达的合适方法。检测基因组改变和 TERT mRNA 表达的结合是检测 TMM 激活的有力策略，可将神经母细胞瘤分为多个临床亚组，用于常规组织病理学实践中的风险分层。
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