*Institute of Pathology, University Hospital of Cologne, Cologne, Germany; †Center for Integrated Oncology Cologne/Bonn, University Hospital of Cologne, Cologne, Germany; ‡Lung Cancer Group Cologne, Department I for Internal Medicine, University Hospital of Cologne, Cologne, Germany; §Labor Dr. Quade und Kollegen GmbH, Cologne, Germany; ‖Institute of Human Genetics, University of Cologne, Cologne, Germany; ¶Targos Molecular Pathology GmbH, Kassel, Germany; #Department of Translational Genomics, University of Cologne, Cologne, Germany; **Cologne Center for Genomics, Cologne, Germany; ††GENterprise GENOMICS GmbH, Mainz, Germany; ‡‡Bristol-Myers Squibb R&D, Princeton, New Jersey; §§Evangelische Kliniken Johanniter, Bonn, Germany; ‖‖Malteser Krankenhaus, Lung Cancer Center, Bonn, Germany; ¶¶Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany; ##Institute for Hematopathology Hamburg, Hamburg, Germany; and ***New Oncology, Cologne, Germany.
J Thorac Oncol. 2015 Jul;10(7):1049-57. doi: 10.1097/JTO.0000000000000570.
The Network Genomic Medicine Lung Cancer was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer performing molecular analyses of more than 3500 patients annually. Because sequential analysis of the relevant driver mutations on fixated samples is challenging in terms of workload, tissue availability, and cost, we established multiplex parallel sequencing in routine diagnostics. The aim was to analyze all therapeutically relevant mutations in lung cancer samples in a high-throughput fashion while significantly reducing turnaround time and amount of input DNA compared with conventional dideoxy sequencing of single polymerase chain reaction amplicons.
In this study, we demonstrate the feasibility of a 102 amplicon multiplex polymerase chain reaction followed by sequencing on an Illumina sequencer on formalin-fixed paraffin-embedded tissue in routine diagnostics. Analysis of a validation cohort of 180 samples showed this approach to require significantly less input material and to be more reliable, robust, and cost-effective than conventional dideoxy sequencing. Subsequently, 2657 lung cancer patients were analyzed.
We observed that comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, we identified driver mutations at the expected prevalence. Furthermore we found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas.
Overall, our data demonstrate the utility of systematic sequencing analysis in a clinical routine setting and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.
网络基因组医学肺癌成立的目的是迅速将科学进展转化为肺癌靶向治疗的早期临床试验，每年对超过 3500 名患者进行分子分析。由于固定样本中相关驱动突变的连续分析在工作量、组织可用性和成本方面具有挑战性，我们在常规诊断中建立了多重平行测序。目的是在高通量的方式下分析肺癌样本中的所有治疗相关突变，同时与传统的单聚合酶链反应扩增子的双脱氧测序相比，显著减少周转时间和输入 DNA 的量。
在这项研究中，我们证明了在常规诊断中使用 102 个扩增子多重聚合酶链反应，然后在 Illumina 测序仪上进行测序在福尔马林固定石蜡包埋组织中的可行性。对 180 个验证队列样本的分析表明，这种方法需要的输入材料明显更少，并且比传统的双脱氧测序更可靠、更稳健、更具成本效益。随后，对 2657 例肺癌患者进行了分析。
我们观察到，综合生物标志物检测除了提供组织学诊断和临床分期外，还提供了新的信息。在连续分析的 2657 例肺癌样本中，我们发现了预期频率的驱动突变。此外，我们还发现了潜在的可靶向 DDR2 突变，在腺癌和鳞状细胞癌中频率为 3%。
总的来说，我们的数据证明了系统测序分析在临床常规环境中的实用性，并强调了这种方法对靶向治疗个别癌症患者的治疗策略的可用性产生的巨大影响。
