靶向RNA测序用于非小细胞肺癌中可操作驱动改变的前期分析

28 March 2022

Targeted RNA sequencing for upfront analysis of actionable driver alterations in non-small cell lung cancer

 (lung cancer; IF:5.705)

• Tan AC, Lai GGY, Tan GS, et al. Utility of incorporating next-generation sequencing (NGS) in an Asian non-small cell lung cancer (NSCLC) population: Incremental yield of actionable alterations and cost-effectiveness analysis. Lung Cancer 2020;139:207-215. (In eng). DOI: 10.1016/j.lungcan.2019.11.022.

• Corresponding author: Sofie Claerhout, Department of Human Genetics, University Hospitals KU Leuven, CDG, 6th floor, Herestraat 49, 3000 Leuven, Belgium. +3216340714, sofie.1.claerhout@uzleuven.be, ORCID: 0000-0003-0857-0211.

OBJECTIVES 目的

Targeted RNA-based Next-Generation Sequencing (tRNA-seq) is increasingly being used in molecular diagnostics for gene fusion detection in non-small cell lung cancer (NSCLC). However, few data support its clinical application for the detection of single nucleotide variants (SNVs) and small insertions/deletions. In this study, we evaluated the performance of tRNA-seq using Archer FusionPlex for simultaneous detection of actionable gene fusions, splice variants, SNVs and indels in formalinfixed, paraffin-embedded NSCLC tissue.

基于靶向RNA的下一代测序(tRNA-seq)正越来越多地用于非小细胞肺癌(NSCLC)基因融合检测的分子诊断。然而，很少有数据支持其在临床应用中检测单核苷酸变异(SNVs)和微小插入/缺失。在本研究中，我们使用Archer FusionPlex来评估tRNA-seq在福尔马林固定、石蜡包埋的NSCLC组织中同时检测可操作的基因融合、剪接变异、SNVs和插入/缺失的性能。

MATERIALS AND METHODS 材料和方法

A total of 126 NSCLC samples, including 20 validation samples and 106 diagnostic cases, were analyzed by targeted DNA-based Next-Generation sequencing (tDNA-seq) followed by tRNA-seq.

共126例NSCLC样本，其中20例为验证样本，106例为诊断样本，采用靶向DNA下一代测序(tDNA-seq)以及靶向RNA下一代测序。

RESULTS 结果

All 28 SNVs and indels in the validation set, and 34 out of 35 mutations in the diagnostic set were identified by tRNA-seq. The only mutation undetected by tRNA-seq, ERBB2 p.(Ser310Tyr), was not included in the current Archer panel design. tRNA-seq revealed one additional BRAF p.(Val600Glu) mutation not found by tDNA-seq. SNVs and indels were correctly called by the vendor supplied software, except for ERBB2 duplication p.(Tyr772_A775dup) which was only detected by an additional in-house developed bio-informatics pipeline. Variant allelic frequency (VAF) values were generally higher at the expression level compared to the genomic level (range 6-96% for tRNA-seq versus 6-61% for tDNA-seq) and low VAF mutations in DNA (6-8% VAF) were all confirmed by tRNA-seq. Finally, tRNA-seq additionally identified a driver fusion or splice variant in 10 diagnostic NSCLC samples including one MET exon 14 skipping variant not detected by tDNA-seq.

验证组的28个单核苷酸变异和插入/缺失，以及诊断组的35个突变中的34个都被tRNA-seq鉴定出来。唯一未被tRNA-seq检测到的突变ERBB2 p.(Ser310Tyr)不包括在当前的Archer面板设计中。tRNA-seq发现了另一个tDNA-seq没有发现的BRAF p.(Val600Glu)突变。供应商提供的软件正确调用了单核苷酸变异和插入/缺失，但ERBB2重复p.(Tyr772_A775dup)除外，该重复p.(Tyr772_A775dup)仅通过另一个内部开发的生物信息学管道检测到。与基因组水平相比，变异等位基因频率(VAF)值在表达水平上普遍较高(tRNA-seq范围为6-96%，tDNA-seq范围为6-61%)，DNA中低VAF突变(6-8% VAF)均被tRNA-seq证实。最后，tRNA-seq在10个诊断性NSCLC样本中还发现了一个驱动融合或剪接变异，包括一个tDNA-seq未检测到的MET第14外显子跳跃变异。

CONCLUSION 结论

Our results demonstrate that tRNA-seq can be implemented in a diagnostic setting as an efficient strategy for simultaneous detection of actionable gene fusions, splice variants, SNVs and indels in NSCLC provided that adequate RNA-seq analysis tools are available, especially for the detection of indels. This approach allows upfront identification of currently recommended targetable molecular alterations in NSCLC samples.

我们的研究结果表明，只要有足够的RNA-seq分析工具，特别是用于检测indel, tRNA-seq可以在诊断环境中作为同时检测NSCLC中可操作的基因融合、剪接变异、单核苷酸变异和插入/缺失的有效策略。这种方法允许提前识别目前推荐的非小细胞肺癌样本中的靶向分子改变。

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