INTRODUCTION Next-generation sequencing (NGS) based on genomic DNA has been widely applied for gene rearrangement detection in patients with non-small cell lung cancer (NSCLC). However, intergenic-breakpoint fusions, in which one or both genomic breakpoints localize to intergenic regions, confound kinase fusion detection. We evaluated the function of intergenic-breakpoint fusions with multiplex molecular testing approaches. METHODS NSCLCs with intergenic-breakpoint fusion identified by DNA-based NGS were analyzed by RNA-based NGS, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). RESULTS Twenty-six cases with single intergenic-breakpoint fusion were identified from a large cohort of NSCLCs using DNA-based NGS. Of the 26 cases, RNA-based NGS detected expressed fusion transcripts in 11 cases, and the genomic breakpoint position did not logically predict breakpoint of the fusion transcript in these cases, possibly due to complex rearrangements (n=5), alternative splicing (n=2) and reciprocal rearrangement (n=4). Nonetheless, no expressed fusion transcript was detected in 5 cases. Moreover, positive ALK IHC was observed in 3 of the remaining 10 cases without RNA-based NGS results. Three intergenic-breakpoint ALK fusion cases with or without RNA-based NGS/IHC confirmation receiving crizotinib treatment showed partial responses. However, one intergenic-breakpoint ROS1 case, given the positive FISH result, received crizotinib but developed progressive disease within one month, possibly owing to no functional fusion transcript detected by RNA-based NGS. CONCLUSIONS Intergenic-breakpoint fusions detected by DNA sequencing confound kinase fusion detection in NSCLC, as functional fusion transcripts may be generated or not. Additional validation testing using RNA/protein assay should be performed in intergenic-breakpoint fusion cases to guide optimal treatment.

中文翻译：

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DNA测序鉴定的基因间断点混淆了非小细胞肺癌中的靶向激酶融合检测

引言 基于基因组 DNA 的二代测序 (NGS) 已广泛应用于非小细胞肺癌 (NSCLC) 患者的基因重排检测。然而，基因间断点融合（其中一个或两个基因组断点位于基因间区域）混淆了激酶融合检测。我们使用多重分子测试方法评估了基因间断点融合的功能。方法采用基于RNA的NGS、免疫组织化学（IHC）和荧光原位杂交（FISH）对DNA-NGS鉴定的基因间断点融合NSCLCs进行分析。结果 使用基于 DNA 的 NGS 从一大群 NSCLC 中鉴定出 26 例具有单一基因间断点融合的病例。在这 26 个病例中，基于 RNA 的 NGS 在 11 个病例中检测到表达的融合转录物，在这些情况下，基因组断点位置在逻辑上无法预测融合转录本的断点，这可能是由于复杂的重排 (n=5)、可变剪接 (n=2) 和相互重排 (n=4)。尽管如此，在 5 个病例中没有检测到表达的融合转录本。此外，在没有基于 RNA 的 NGS 结果的其余 10 例中，有 3 例观察到阳性 ALK IHC。接受克唑替尼治疗的三个基因间断点 ALK 融合病例有或没有基于 RNA 的 NGS/IHC 确认显示部分反应。然而，鉴于 FISH 结果呈阳性，一例基因间断点 ROS1 病例接受了克唑替尼治疗，但在 1 个月内出现疾病进展，这可能是由于基于 RNA 的 NGS 未检测到功能性融合转录本。结论 DNA 测序检测到的基因间断点融合混淆了 NSCLC 中的激酶融合检测，因为可能会产生或不产生功能性融合转录本。应在基因间断点融合病例中使用 RNA/蛋白质测定进行额外的验证测试，以指导最佳治疗。