From the Division of Cytogenetic and Molecular Pathology, ProPath, Dallas, Texas (Bridge).
the Department of Pathology & Microbiology, University of Nebraska Medical Center, Omaha (Bridge).
Arch Pathol Lab Med. 2024 May 1;148(5):538-544. doi: 10.5858/arpa.2023-0047-CP.
Next-generation sequencing-based approaches using RNA have increasingly been used by clinical laboratories for the detection of fusion genes, intragenic rearrangements, and exon-skipping events. Correspondingly, the College of American Pathologists (CAP) has advanced RNA sequencing proficiency testing (PT) to ensure optimal performance of these assays.
To report on laboratory performance and practices of RNA sequencing for the detection of fusion genes, intragenic rearrangements, and exon-skipping events using CAP PT data from 8 mailings (2018-A through 2021-B).
CAP PT RNA sequencing program results from 153 laboratories across 24 proficiency test specimens, interrogating 22 distinct engineered fusion transcripts, were analyzed for correct identification of the fusion event, associated performance variables, and laboratory practices.
Overall, the 4-year program detection rate (sensitivity) was 95.5% (1486 of 1556 results). False-negative rates were 3.6% (53 of 1463) and 18.3% (17 of 93) for fusion gene and intragenic rearrangement/exon-skipping events, respectively. Only 19 false-positive results were reported among the 8 PT mailings, and most were likely the result of preanalytical or postanalytical errors. There were no practice characteristics (eg, instrumentation, sequencing method) significantly associated with the fusion detection results.
These data reveal a high overall sensitivity and specificity for fusion gene detection by participating laboratories using clinical RNA sequencing. Performance was comparable across all laboratories, regardless of methodology. The fraction of false-negative results for intragenic rearrangement/exon-skipping events was greater than that for the chimeric fusion genes. False-negative results could not be attributed to any specific practice characteristics.
临床实验室越来越多地采用基于下一代测序的 RNA 方法来检测融合基因、基因内重排和外显子跳跃事件。相应地，美国病理学家学院 (CAP) 已经推进了 RNA 测序能力验证 (PT)，以确保这些检测的最佳性能。
报告使用 CAP PT 数据（共 8 次邮寄，分别为 2018-A 至 2021-B）进行 RNA 测序以检测融合基因、基因内重排和外显子跳跃事件的实验室性能和实践情况。
对来自 24 个能力验证标本的 153 个实验室的 CAP PT RNA 测序计划结果进行分析，这些标本检测了 22 种不同的工程融合转录本，以确定融合事件的正确识别、相关性能变量和实验室实践情况。
总体而言，该 4 年计划的检测率（灵敏度）为 95.5%（1486 次检测中有 1463 次正确）。融合基因和基因内重排/外显子跳跃事件的假阴性率分别为 3.6%（53 次中有 53 次）和 18.3%（93 次中有 17 次）。在 8 次能力验证邮寄中，仅报告了 19 次假阳性结果，其中大多数可能是由于分析前或分析后错误。没有发现与融合检测结果显著相关的实践特征（例如，仪器、测序方法）。
这些数据显示，参与实验室使用临床 RNA 测序进行融合基因检测具有较高的总体灵敏度和特异性。所有实验室的性能相当，无论方法如何。基因内重排/外显子跳跃事件的假阴性率大于嵌合融合基因的假阴性率。假阴性结果不能归因于任何特定的实践特征。
