Department of Pathology and Laboratory Medicine, Molecular Diagnostic Service, Memorial Sloan Kettering Cancer Center, New York, New York.
Thoracic Oncology Service, Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
J Mol Diagn. 2022 Jun;24(6):642-654. doi: 10.1016/j.jmoldx.2022.03.006. Epub 2022 Apr 14.
Several kinase fusions are established targetable drivers in lung cancers. However, rapid and comprehensive detection remains challenging because of diverse partner genes and breakpoints. We assess the clinical utility and performance of a rapid microfluidic multiplex real-time PCR-based assay for simultaneous query of fusions involving ALK, ROS1, RET, and NTRK1/2/3, as well as MET exon 14 skipping, using a 3-hour automated process. Dual analytic strategies were utilized: fusion-specific amplification and 3' to 5' expression imbalance. One-hundred and forty-three independent, formalin-fixed, paraffin-embedded tumor samples (112 surgical specimens, 31 cytologic cell blocks) were analyzed: 133 with known kinase gene alterations and 10 negative samples based on clinically validated next-generation sequencing. Testing was successful in 142 (99%) cases. The assay demonstrated a sensitivity of 97% (28/29), 100% (31/31), 92% (22/24), 81% (22/27), and 100% (20/20) for ALK, RET, ROS1, and NTRK1/2/3 rearrangements and MET exon 14 skipping alterations, respectively, with 100% specificity for all. Concordant results were achieved in specimens aged up to 5 years, with >10% tumor, and inputs of at least 9 mm (surgical specimens) and 9000 cells (cytologic cell blocks). The assay enables rapid screening for clinically actionable kinase alterations with quicker turnaround and lower tissue requirements compared with immunohistochemistry and molecular methods, while also circumventing the infrastructure dependencies associated with next-generation sequencing and fluorescence in situ hybridization.
几种激酶融合是肺癌中已确定的可靶向驱动因素。然而，由于不同的伙伴基因和断点，快速全面的检测仍然具有挑战性。我们评估了一种快速微流控多重实时 PCR 检测方法的临床实用性和性能，该方法可同时检测涉及 ALK、ROS1、RET 和 NTRK1/2/3 的融合，以及 MET 外显子 14 跳跃，采用 3 小时自动化处理。使用两种分析策略：融合特异性扩增和 3' 到 5' 表达失衡。对 143 个独立的福尔马林固定石蜡包埋肿瘤样本（112 个手术标本，31 个细胞学细胞块）进行了分析：133 个具有已知激酶基因改变，10 个为基于临床验证的下一代测序的阴性样本。在 142 例（99%）病例中检测成功。该检测方法对 ALK、RET、ROS1 和 NTRK1/2/3 重排以及 MET 外显子 14 跳跃改变的灵敏度分别为 97%（28/29）、100%（31/31）、92%（22/24）、81%（22/27）和 100%（20/20），特异性为 100%。在年龄高达 5 年的标本中，肿瘤含量>10%，输入量至少为 9mm（手术标本）和 9000 个细胞（细胞学细胞块）时，均可获得一致的结果。与免疫组织化学和分子方法相比，该检测方法可快速筛选出具有临床可操作性的激酶改变，检测周转时间更短，组织需求更少，同时还避免了与下一代测序和荧光原位杂交相关的基础设施依赖。
