Division of Pulmonary and Critical Care Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
Institute of Pulmonary Diseases, Sun Yat-sen University, Guangzhou, China.
Mol Diagn Ther. 2021 Jul;25(4):487-494. doi: 10.1007/s40291-021-00532-8. Epub 2021 Jun 16.
Precise detection of anaplastic lymphoma kinase (ALK) rearrangement guides the application of ALK-targeted tyrosine kinase inhibitors (ALK-TKIs) in patients with non-small-cell lung cancer (NSCLC). Next-generation sequencing (NGS) has been widely used in clinics, but DNA-based NGS used to detect fusion genes has delivered false-negative results. However, fusion genes can be successfully detected at the transcription level and with higher sensitivity using RNA-based reverse transcription polymerase chain reaction (RT-PCR).
This study compared the performance of RT-PCR and NGS in the detection of echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion in Chinese patients with NSCLC.
Formalin-fixed paraffin-embedded tissues from 153 patients who were pathologically diagnosed as having NSCLC were collected from November 2017 to October 2019. Both DNA/RNA-based NGS and RNA-based RT-PCR were used to detect EML4-ALK fusion. For samples with discordant ALK status results, fluorescence in situ hybridization (FISH) or Sanger sequencing was used to further confirm the ALK status.
In total, 124 samples were successfully analyzed using both NGS and RT-PCR. For 118 samples, results were consistent between NGS and RT-PCR, with 25 reported as ALK fusion positive and 93 as ALK fusion negative, achieving a concordance rate of 95.16%. Among the six samples with disconcordant results, five were positive using RT-PCR but negative using NGS, and one was positive using NGS but negative using RT-PCR. Four of six cases with disconcordant results (three RT-PCR positive and one NGS positive) were successfully validated using either FISH or Sanger sequencing.
Compared with NGS, RT-PCR appears to be a reliable method of detecting EML4-ALK fusion in patients with NSCLC.
精确检测间变性淋巴瘤激酶（ALK）重排可指导非小细胞肺癌（NSCLC）患者应用 ALK 靶向酪氨酸激酶抑制剂（ALK-TKI）。下一代测序（NGS）已广泛应用于临床，但用于检测融合基因的基于 DNA 的 NGS 曾报告假阴性结果。然而，基于 RNA 的逆转录聚合酶链反应（RT-PCR）可在转录水平以更高的灵敏度成功检测融合基因。
本研究比较了 RT-PCR 和 NGS 在中国 NSCLC 患者中检测棘皮动物微管相关蛋白样 4（EML4）-ALK 融合的性能。
收集 2017 年 11 月至 2019 年 10 月病理诊断为 NSCLC 的 153 例患者的福尔马林固定石蜡包埋组织。采用基于 DNA/RNA 的 NGS 和基于 RNA 的 RT-PCR 检测 EML4-ALK 融合。对于 ALK 状态检测结果不一致的样本，采用荧光原位杂交（FISH）或 Sanger 测序进一步确认 ALK 状态。
共对 124 例样本同时进行了 NGS 和 RT-PCR 分析。118 例样本的 NGS 和 RT-PCR 结果一致，25 例报告为 ALK 融合阳性，93 例为 ALK 融合阴性，一致性率为 95.16%。6 例结果不一致的样本中，5 例 RT-PCR 阳性而 NGS 阴性，1 例 NGS 阳性而 RT-PCR 阴性。6 例结果不一致的样本中有 4 例（3 例 RT-PCR 阳性，1 例 NGS 阳性）通过 FISH 或 Sanger 测序成功验证。
与 NGS 相比，RT-PCR 似乎是一种可靠的 NSCLC 患者 EML4-ALK 融合检测方法。
