Department of Pathology, Division of Pathology, University of Colorado-Anschutz Medical Campus, Aurora, Colorado.
Department of Pathology, Division of Pathology, University of Colorado-Anschutz Medical Campus, Aurora, Colorado.
J Thorac Oncol. 2019 Apr;14(4):737-741. doi: 10.1016/j.jtho.2018.12.020. Epub 2019 Jan 9.
Genomic variants that lead to MET proto-oncogenem receptor tyrosine kinase (MET) exon 14 skipping represent a potential targetable molecular abnormality in NSCLC. Consequently, reliable molecular diagnostic approaches that detect these variants are vital for patient care.
We screened tumor samples from patients with NSCLC for MET exon 14 skipping by using two distinct approaches: a DNA-based next-generation sequencing assay that uses an amplicon-mediated target enrichment and an RNA-based next-generation sequencing assay that uses anchored multiplex polymerase chain reaction for target enrichment.
The DNA-based approach detected MET exon 14 skipping variants in 11 of 856 NSCLC samples (1.3%). The RNA-based approach detected MET exon 14 skipping in 17 of 404 samples (4.2%), which was a statistically significant increase compared with the DNA-based assay. Among 286 samples tested by both assays, RNA-based testing detected 10 positives, six of which were not detected by the DNA-based assay. Examination of primer binding sites in the DNA-based assay in comparison with published MET exon 14 skipping variants revealed genomic deletion involving primer binding sequences as the likely cause of false negatives. Two samples positive via the DNA-based approach were uninformative via the RNA-based approach due to poor-quality RNA.
By circumventing an inherent limitation of DNA-based amplicon-mediated testing, RNA-based analysis detected a higher proportion of MET exon 14 skipping cases. However, RNA-based analysis was highly reliant on RNA quality, which can be suboptimal in some clinical samples.
导致 MET 原癌基因受体酪氨酸激酶（MET）外显子 14 跳跃的基因组变异代表了 NSCLC 中一个潜在的可靶向分子异常。因此，可靠的分子诊断方法对于患者的治疗至关重要。
我们使用两种不同的方法筛选了 NSCLC 患者的肿瘤样本，以检测 MET 外显子 14 跳跃：一种是基于 DNA 的下一代测序检测方法，该方法使用扩增子介导的靶向富集；另一种是基于 RNA 的下一代测序检测方法，该方法使用锚定多重聚合酶链反应进行靶向富集。
基于 DNA 的方法在 856 个 NSCLC 样本中的 11 个（1.3%）中检测到 MET 外显子 14 跳跃变体。基于 RNA 的方法在 404 个样本中的 17 个（4.2%）中检测到 MET 外显子 14 跳跃，与基于 DNA 的检测方法相比，这是一个统计学上的显著增加。在两种检测方法都检测的 286 个样本中，RNA 检测到 10 个阳性，其中 6 个未被 DNA 检测到。对 DNA 检测方法中的引物结合位点进行检查，并与已发表的 MET 外显子 14 跳跃变异体进行比较，结果显示可能是由于基因组缺失涉及引物结合序列，导致假阴性。通过 DNA 方法检测为阳性的两个样本，由于 RNA 质量较差，通过 RNA 方法检测为无意义。
通过规避基于 DNA 的扩增子介导检测的固有限制，基于 RNA 的分析检测到更高比例的 MET 外显子 14 跳跃病例。然而，RNA 分析高度依赖于 RNA 质量，在某些临床样本中可能不理想。
