用于检测实体瘤中基因扩增及其他基因扩增的基于扩增子的下一代测序检测方法的分析验证和性能评估
Analytical Validation and Performance Evaluation of Amplicon-Based Next-Generation Sequencing Assays for Detecting and Other Gene Amplifications in Solid Tumors.
作者信息
Olkhov-Mitsel Ekaterina, Chan Danny, Craddock Kenneth J, Lin August, Luk Grace, Goswami Rashmi S, Wang Hong, Plotkin Anna, Nofech-Mozes Sharon, Hwang David M, Huang Weei-Yuarn
机构信息
Precision Diagnostics and Therapeutics Program, Division of Anatomic Pathology, Department of Laboratory Medicine and Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, ON M4N 3M5, Canada.
Department of Laboratory Medicine and Pathobiology, Temerty Faculty of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada.
出版信息
Cancers (Basel). 2024 Nov 23;16(23):3927. doi: 10.3390/cancers16233927.
BACKGROUND
Targeted next-generation sequencing (NGS) panels are increasingly being utilized to identify actionable gene amplifications (copy number > 4) among solid tumors.
METHODS
This study validated the analytical performance of two amplicon-based NGS assays, the Oncomine Comprehensive Panel (OCAv3) and the Oncomine Focus Assay (OFA), for detecting gene amplification in formalin-fixed paraffin-embedded (FFPE) tumors of varying cellularity. OCAv3 was assessed for amplification detection in 756 FFPE samples comprising various tumor types.
RESULTS
We demonstrated that with standardized quality control metrics, including median absolute pairwise difference score, these assays can achieve a near-perfect positive predictive value, although their sensitivity for detecting amplifications significantly decreased in tumors with cellularity below 30%. Stratifying tumor cellularity into 10-30%, 31-60%, and 61-95% groups revealed significantly higher gene amplification detection rates in the 31-60% and 61-95% groups the 10-30% group (20.6% and 26.7% vs. 9.2%, < 0.0001). When considering all detected gene amplifications, the average amplification calling per sample was nearly five-fold lower in the 10-30% group the 61-95% group (0.11 vs. 0.52; < 0.0001). To further investigate the analytic performance of OCAv3 in detecting amplification, we analyzed a cohort of 121 uterine carcinomas with confirmed status by HER2 IHC or FISH, in which a threshold incorporating amplifications and tumor cellularity achieved 79% sensitivity and 100% specificity, potentially eliminating the need for FISH analysis in 34% of equivocal cases. In a separate validation cohort, similar analytical performance was observed, with the threshold demonstrating consistent sensitivity and specificity.
CONCLUSIONS
This study highlights the strengths and limitations of amplicon-based NGS assays in detecting amplifications using real-world data.
背景
靶向新一代测序（NGS）面板越来越多地用于识别实体瘤中可操作的基因扩增（拷贝数>4）。
方法
本研究验证了两种基于扩增子的NGS检测方法，即安捷伦全基因组癌症panel（OCAv3）和安捷伦癌症热点panel（OFA），在检测不同细胞密度的福尔马林固定石蜡包埋（FFPE）肿瘤中基因扩增的分析性能。对756份包含各种肿瘤类型的FFPE样本评估了OCAv3的扩增检测情况。
结果
我们证明，通过标准化的质量控制指标，包括中位绝对成对差异评分，这些检测方法可以实现近乎完美的阳性预测值，尽管它们在细胞密度低于30%的肿瘤中检测扩增的灵敏度显著降低。将肿瘤细胞密度分为10 - 30%、31 - 60%和61 - 95%组后发现，31 - 60%组和61 - 95%组的基因扩增检测率显著高于10 - 30%组（分别为20.6%和26.7%，而10 - 30%组为9.2%，P<0.0001）。在考虑所有检测到的基因扩增时，10 - 30%组每个样本的平均扩增检出率比61 - 95%组低近五倍（分别为0.11和0.52；P<0.0001）。为了进一步研究OCAv3在检测HER2扩增方面的分析性能，我们分析了一组121例经HER2免疫组化（IHC）或荧光原位杂交（FISH）确诊HER2状态的子宫癌，其中结合扩增和肿瘤细胞密度的阈值实现了79%的灵敏度和100%的特异性，在34%的可疑病例中可能无需进行FISH分析。在另一个验证队列中，观察到了类似的分析性能，该阈值显示出一致的灵敏度和特异性。
结论
本研究强调了基于扩增子的NGS检测方法在利用实际数据检测扩增方面的优势和局限性。
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