基于扩增子的超快速二代测序在非鳞状非小细胞肺癌中的应用
1Laboratory of Clinical and Experimental Pathology, Centre Hospitalier Universitaire de Nice,Université Côte d’Azur, CHU de Nice, 2Hospital-Integrated Biobank (BB-0033-00025),Université Côte d’Azur, Hôpital Pasteur, CHU de Nice, 3Institut Hospitalo-Universitaire (IHU), RespirERA,Université Côte d’Azur, Hôpital Pasteur, CHU de Nice, 4FHU OncoAge,Université Côte d’Azur, 5Team 4, Institute of Research on Cancer and Aging (IRCAN), CNRS INSERM, Centre Antoine-Lacassagne,Université Côte d’Azur, 6Department of Pulmonary Medicine and Thoracic Oncology, Centre Hospitalier Universitaire de Nice,Université Côte d’Azur
Summary
用于非鳞状非小细胞肺癌 （NS-NSCLC） 护理管理的分子生物标志物的增加促使了快速可靠的分子检测方法的发展。我们描述了使用超快速下一代测序 （NGS） 方法对 NS-NSCLC 患者进行基因组改变评估的工作流程。
Abstract
在过去几年中，用于非鳞状非小细胞肺癌 （NS-NSCLC） 患者靶向治疗的分子改变数量显着增加。对于晚期或转移性NS-NSCLC患者的最佳护理，必须检测分子异常，从而可以进行靶向治疗，从而改善总生存期。然而，这些肿瘤会产生耐药机制，这些机制可能使用新疗法靶向。一些分子改变也可以调节治疗反应。NS-NSCLC的分子表征必须在短时间内完成，在不到10个工作日的时间内完成，这是国际指南所建议的。此外，用于基因组分析的组织活检的来源是多种多样的，并且随着侵入性较小的方法和方案的发展，其尺寸不断减小。因此，病理学家面临的挑战是执行有效的分子技术，同时保持高效和快速的诊断策略。在这里，我们描述了基于超快速扩增子的下一代测序 （NGS） 工作流程，用于诊断 NS-NSCLC 患者的日常实践。我们发现，该系统能够在适当的TAT中识别当前用于胸部肿瘤学精准医学的分子靶点。
Introduction
在过去十年中，靶向和免疫疗法的发展显著提高了非鳞状非小细胞肺癌 （NS-NSCLC） 的总生存期 （OS）1,2。在这方面，在过去几年中，治疗NS-NSCLC时需要分析的强制性基因和分子靶点的数量有所增加3,4。
目前的国际指南建议在诊断晚期 NS-NSCLC5 时检测 EGFR、ALK、ROS1、BRAF、NTRK、RET 和 MET。此外，由于新药最近在临床试验中取得了非常有希望的结果，因此不久将在许多其他基因中筛选出额外的基因组改变，特别是KRAS和HER2，以及BRAC1/BRAC2，PI3KA，NRG1和NUT 6,7,8,9。此外，不同相关基因（如 STK11、KEAP1 和 TP53）的状态可能有助于更好地预测对某些靶向治疗和/或免疫检查点抑制剂 （ICI） 的反应或耐药性10,11,12。
重要的是，必须立即报告分子改变，以确保谨慎的临床决策。缺乏肿瘤的分子特征可能导致开始非靶向治疗，例如化疗联合/不联合免疫治疗，导致次优治疗策略，因为化疗反应在具有可操作改变的患者中受到限制，例如 EGFR 突变或基因融合13。
此外，目前在新辅助和/或辅助治疗中靶向治疗/免疫疗法的发展可能导致系统地寻找，至少在早期NS-NSCLC中寻找EGFR和ALK的改变，因为ICI应该只在EGFR和ALK14的野生型肿瘤中给药。由于奥希替尼（第三代EGFR酪氨酸激酶抑制剂）可用作EGFR突变NS-NSCLC的辅助治疗15，因此现在还必须检测早期NS-NSCLC中是否存在EGFR突变。
评估不同生物标志物以预测NS-NSCLC患者对不同靶向治疗和/或免疫治疗的反应的策略正在快速发展，这使得这些生物标志物的顺序识别变得困难3,16。在这方面，下一代测序 （NGS） 现在是 NS-NSCLC 5,17 中基因改变的高通量并行评估的最佳方法。
然而，NGS 工作流程可能难以掌握，并且可能需要更长的 TAT18,19。因此，许多中心仍然采用序贯方法（免疫组化 （IHC）、荧光原位杂交 （FISH） 和/或靶向测序）。然而，在样本量小的情况下，这种策略是有限的，最重要的是，因为在NS-NSCLC20中需要测试的可操作突变数量增加。因此，允许快速评估基因改变的超快速和直接的检测方法对于最佳临床决策变得越来越重要。此外，经批准和认可的分子检测系统正成为特定靶向治疗处方的强制性要求。
在这里，我们描述了一种用于NS-NSCLC分子检测的超快速和自动化的基于扩增子的DNA/RNA NGS测定，该测定用于法国尼斯大学医院临床和实验病理学实验室（LPCE），并根据ISO 15189标准获得法国认可委员会（COFRAC）（https://www.cofrac.fr/）的认可。COFRAC 证明该实验室符合标准 ISO 15189 和 COFRAC 应用规则的要求，即在实验室执行的测序仪上对自动 NGS 分子分析中的测试/校准活动进行测试/校准。根据公认的国际标准 ISO 15189 的认证证明了实验室在规定范围内的技术能力以及该实验室中适当管理体系的正确运行。讨论了该工作流程的优点和局限性，从组织活检样本的制备到获得报告。
Protocol
所有程序均已获得当地伦理委员会（人类研究伦理委员会，尼斯大学医院中心，Tumorothèque BB-0033-00025）的批准。获得所有患者的知情同意，使用样本和生成的数据。所有样本均来自2022年9月20日至1月31日期间在LPCE（法国尼斯）诊断为NS-NSCLC的患者，作为医疗护理的一部分。 1. 使用自动纯化仪器（API）制备FFPE DNA和RNA样品（处理时间：5小时15分钟） 在仪器上?…
Representative Results
使用我们最近的出版物21 中详细描述的此处介绍的程序，我们开发了一种最佳工作流程，用于评估分子改变，作为常规临床实践中的反射测试，用于使用基于超快速扩增子的下一代测序方法诊断 NS-NSCLC 患者。该方法的分子工作流程如 图1所示。该组合中包含的基因列表如 补充图1所示。 对259例NS-NSCLC患者进行了分子分析。?…
Discussion
开发一种基于超快速扩增子的 NGS 方法作为反射测试，用于诊断任何阶段的 NS-NSLC 的分子改变评估，是检测 NS-NSCLC中所有指南推荐和新兴生物标志物的最佳选择5,22,23。虽然序贯方法（IHC、PCR、FISH）仅关注特定基因并可能导致组织材料耗尽，但这种 NGS 工作流程允许对 50 个基因的状态进行特异性和灵敏的评估，包括突变、融合?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢赛默飞世尔科技为我们提供了使用其设备和材料的可能性。
Materials
| 96 well hard shell plate clear | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | 4483354 | |
| Adhesive PCR Plate Foil | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | AB0626 | |
| AutoLys M tube | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A38738 | FFPE sample processing tubes |
| Genexus Barcodes 1-32 HD | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40261 | |
| Genexus GX5 Chip and Genexus Coupler | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40269 | |
| Genexus Pipette Tips | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40266 | |
| Genexus Purification Instrument | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A48148 | Automated purification instrument (API) |
| Genexus Sequencing Kit | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40271 | |
| Genexus Templating Strips 3-GX5 and 4 | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A40263 | |
| Genexus Integrated Sequencer | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A45727 | |
| Ion Torrent Genexus FFPE DNA/RNA Purification Combo Kit | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A45539 | |
| Oncomine Precision Assay GX (OPA) Panel (included Strips 1 and 2-HD) | Thermo Fisher Scientific (Waltham, Massachusetts, USA) | A46291 |
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Cite This Article
Bontoux, C., Lespinet-Fabre, V., Bordone, O., Tanga, V., Allegra, M., Salah, M., Lalvée, S., Goffinet, S., Benzaquen, J., Marquette, C., Ilié, M., Hofman, V., Hofman, P. Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer. J. Vis. Exp. (199), e65190, doi:10.3791/65190 (2023).