Department of Genetics and Clinical Immunology, The Institute of Tuberculosis and Lung Diseases, 01-138 Warsaw, Poland.
Department of Pathology, The Institute of Tuberculosis and Lung Diseases, 01-138 Warsaw, Poland.
Int J Mol Sci. 2024 Jul 19;25(14):7908. doi: 10.3390/ijms25147908.
The emergence of targeted therapies in non-small-cell lung cancer (NSCLC), including inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, has increased the need for robust companion diagnostic tests. Nowadays, detection of actionable variants in exons 18-21 of the gene by qPCR and direct DNA sequencing is often replaced by next-generation sequencing (NGS). In this study, we evaluated the diagnostic usefulness of targeted NGS for druggable variants testing in clinical NSCLC material previously analyzed by the IVD-certified qPCR test with respect to DNA reference material. We tested 59 NSCLC tissue and cytology specimens for variants using the NGS 'TruSight Tumor 15' assay (Illumina) and the qPCR 'cobas EGFR mutation test v2' (Roche Diagnostics). The sensitivity and specificity of targeted NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of variants. NGS demonstrated a sufficient lower detection limit for diagnostic applications (VAF < 5%) in DNA reference material; all variants were correctly identified. NGS showed high repeatability of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 76.14% (Cohen's Kappa = 0.5933). The majority of discordant results concerned false-positive detection of exon 20 insertions by qPCR. A total of 9 out of 59 (15%) clinical samples showed discordant results for one or more variants in both assays. Additionally, we observed to be a frequently co-mutated gene in -positive NSCLC patients. In conclusion, targeted NGS showed a number of superior features over qPCR in variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity), which might enhance the basic diagnostic report.
在非小细胞肺癌（NSCLC）中，靶向治疗的出现，包括表皮生长因子受体（EGFR）酪氨酸激酶抑制剂的出现，增加了对强大伴随诊断测试的需求。如今，通过 qPCR 和直接 DNA 测序检测基因外显子 18-21 中的可操作变异体，通常被下一代测序（NGS）所取代。在这项研究中，我们评估了针对 NSCLC 临床标本中可靶向治疗的 NGS 检测在 DNA 参考物质方面对先前经 IVD 认证的 qPCR 测试分析的 druggable 变异体检测的诊断有用性。我们使用 NGS 'TruSight Tumor 15' 测定法（Illumina）和 qPCR 'cobas EGFR mutation test v2'（罗氏诊断）对 59 个 NSCLC 组织和细胞学标本进行了 变体检测。使用具有已知变异等位基因频率（VAF）的生物合成和生物 DNA 参考物质评估了靶向 NGS 检测的敏感性和特异性。在 DNA 参考物质中，NGS 显示出足够低的诊断应用检测下限（VAF <5%）；所有变异体均被正确识别。在运行之间，NGS 显示出 VAF 评估的高可重复性（CV% 为 0.02 至 3.98）。在临床标本中，NGS 和 qPCR 之间的总体一致性为 76.14%（Cohen's Kappa = 0.5933）。大多数不一致的结果涉及 qPCR 对 外显子 20 插入的假阳性检测。在两个检测中，总共 59 个临床样本中有 9 个（15%）显示一个或多个 变体的不一致结果。此外，我们观察到 在 -阳性 NSCLC 患者中是一个经常共突变的基因。总之，与 qPCR 相比，靶向 NGS 在变异体检测方面具有许多优势（变异体的准确识别、等位基因频率的计算、高分析灵敏度），这可能会增强基本诊断报告。
